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Annu. Rev. Biochem. Copyright
© 1992
1992. 61:283-306
by Annual Reviews Inc. All rights reserved
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
CHROMOSOME AND PLASMID PARTITION IN ESCHERICHIA COLI Sofa Hiraga Department of Molecular Genetics, Institute for Medical Genetics, Kumamoto University Medical School, Kumamoto 862, Japan KEY WORDS:
chromosome partition, plasmid partition, MukB protein, ATPase. filamentous
protein polymer
CONTENTS INTRODUCTION.....................................................................................
283
ANALYSIS OF GENES INVOLVED IN CHROMOSOME PARTITION ..... ...........
286 286 286
Two Categories of Mechanisms. . . ... . . . .. . . . . ........... ....... . . . . ... . . . . . . . . . . . . . ..... . . . . . . Bacterial par Mutants Defective in Topoisomerases .... muk Mutants; Mutants that Produce Chromosome-less Cells......... ........ ........... THE mukB GENE AND ITS PRODUCT........................................................ Properties of the mukB Mutants......................... .............................. ........
Predicted Secondary Structure of MukB Protein....... .. .......... ..... . . . . . . . ........ . . . . Properties of MukB Protein in vitro. ...... . . .... . . . . . .... ........... . . . .. . . .. . . . . . . . . . . . .. . . . Filamentous Protein Polymers in E. coli.................................................... PARTITION MECHANISM OF PLASMlDS . ... ...... ............................... .......... Plasmid-Encoded Proteins and the cis-Acting Region of F and PI
Plasmids Essential for Partition..................................................
Partition Genes of the lncFfl Plasmids Rl and NRI ................. .... . . .... . . . . . . . . ... The cis-Acting par Site of pSCIOI Plasmid.............. ....................
Partition of a Mini-F Plasmid in a mukB-Disrupted Null Mutant......................
CONCLUDING REMARKS................................ . ... ... . .... ... . ..................
288 290 290 291
293
294 295 295
301
302 302 303
INTRODUCTION In bacteria, chromosomal DNA is replicated and daughter chromosome mole cules are accurately partitioned into daughter cells prior to cell division. 0066-4154/92/0701-0283$02.00
283
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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Daughter cells almost always receive at least one copy of the chromosome; anucleate (chromosome-less) cells account for less than 0.03 % of the popula tion in growing cultures of wild-type strains of Escherichia coli. Before septation at midcell, daughter chromosome molecules are separated from each other and located at the cell quarter positions (1/4 and 3/4 cell lengths), where the daughter chromosomes appear more compact than replicating chromo somes. Jacob et al (1) proposed a model in which the partitioning of replicated chromosomes and molecules of F plasmid (the classical E. coli sex factor) into daughter cells involves a physical connection between a daughter DNA strand and part of the cell envelope, and in which the driving force for segregation of the two daughter chromosomes and of F plasmids must be provided by insertion of new cell envelope material at midcell, i.e. between two attachment sites. A ccording to one version of this model, the chromo some replication origin oriC or its flanking region is the specific site that is attached to the cell envelope, and daughter chromosomes move to opposite directions by elongation of the envelope, in concert with the progress of chromosome replication. However, experimental results showed that growth of the cell wall takes place by random insertion over the entire surface during most of the E. coli cell cycle, although during septum formation peptidogly can precursors are inserted preferentially at midcell (2-4). A lthough biochemical data showed temporary association of the bacterial membrane with the oriC regions of the bacterial chromosome and with oriC plasmids (minichromosomes) (for example, 5-7), it is doubtful that the membrane attachment of the oriC region actually plays an essential role in partition of the chromosome and of oriC plasmids to daughter cells in vivo. In the absence of selective pressure, oriC plasmids are unstably maintained in a population of actively dividing bacterial cells, and are lost rapidly from the bacterial population. They do not have a partition mechanism, but are parti tioned essentially at random into daughter cells. By contrast, an oriC plasmid earrying the sopA, sopB, and sopC genes (parA, parB, and parC) of the F plasmid is stably maintained in spite of the low copy number of plasmid molecules per cell (8). Therefore it is unlikely that oriC and/or its flanking regions play an essential role in chromosome partitioning. The two strands of the double-stranded chromosomal DNA in a newborn cell can be distinguished by the fact that one strand (the younger strand) was formed during the most recent round of replication and the other (the older strand) was formed during some earlier round of replication. Upon replica tion, the younger and older chromosomes separate from one another to eventually reside within the complementary new daughter cells. The direc tions in which the new chromosomes are partitioned can also be easily discriminated by the fact that every cell has a younger polar cap, formed at the
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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previous division, and an older polar cap formed during some earlier division. Does the cell distinguish between the two daughter chromosomes in conjunc tion with the direction of partition? To answer the question , studies have been made of the partitioning of younger and older daughter chromosomes into daughter cells of E. coli during division, and it has been shown that there is a preferental segregation of the older chromosome towards the cell with the older polar cap (for a review, see 9). Helmstetter & Leonard (10 ) propose that the cell envelope contains a large number of sites capable of binding to the chromosomal replication origin, oriC, that a polymerizing DNA strand be comes attached to one of the sites at initiation of a round of replication, and that the attachment sites are distributed throughout the actively growing cell envelope, i. e. lateral envelope and septum, but not in the existing cell poles. This asymmetric distribution of oriC attachment sites could account for the experimentally observed nonrandom segregation of the older daughter chromosome with cell strain and growth rate. The multisite attachment con cept could also account for at least some of the instability with which minichromosomes are maintained (9). It has been found that the "positioning" of daughter chromosome molecules at the cell quarter pos itio ns req uir es po stre pli ca tion al protein synthesis (1 1 , 1 2; for a review, see 1 3 ). When protein synthesis is inhibited by starvation for amino acids or by the addition of an inhibitor of protein synthesis, a replicat ing chromosome completes its replication and the r esulting daughter chromo somes remain at the midcell, close to each other. When protein synthesis resumes by the addition of the amino acids or by removal of the inhibitor, the daughter chromosomes can rapidly move from midcell to the cell quarter positions before a detectable increase in cell lcngth occurs (1 2). This suggests that daughter chromosomes are transported by an unknown mechanism, but not by elongation of the cell envelope itself. The positioning of daughter chromosomes from midcell to cell quarter positions after resumption of protein synthesis is achieved even in the presence of an inhibitor of DNA gyrase, nalidixic acid (50 JLg/ml), or novobiocin (l00 JLg/ml). Moreover, it was found that the positioning after resumption of protein synthesis is achiev ed even when the initiation of chromosome replication is inhibit ed using a temperature-sensitive dnaC mutant, which is defective in initiation of chromosome replication at a nonpermissive temperature. Thus the reinitiation of chromosome r eplic atio n is not required for the positioning of dau ghter chromosomes. The results described above strongly indicate that E. coli has a mechanism, albeit unknown, for positioning daughter chromosomes. This mechanism may be promoted by events that are dependent on pos trep lic ation al protein synthesis (1 2). The events may also promote septum formation, causing the cell to enter the D (division) period from the C period (for a review, see 1 4). Individual chromosomes are fixed in position within growing ,
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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cells both before and during replication, but they are rapidly moved apart by a fixed distance (unit length) immediately after replication has been completed (15). Understanding how daughter chromosomes are positioned at cell quarter positions before cell division will provide insights into the fundamental process of partition. To analyze the molecular mechanism of chromosome positioning, isolation and characterization of mutants defective in this mech anism are advantageous. Techniques have been developed by us to isolate mutants that produce, upon cell division and at a non-negligible frequency, one anucleate daughter cell of normal cell sizc and one nucleate daughter cell carrying two copies of daughter chromosomes (16). Many mutants have been isolated by using these techniques. Our recent results indicate that a newly discovered gene, mukB, is involved in chromosome positioning at the cell quarter positions. The amino acid sequence deduced from the nucleotide sequence of the cloned mukB gene and the properties of the purified MukB protein suggest that the MukB protein is the first candidate found for a force-generating (mechanochemical) enzyme in bacteria (17, 18). The mech anism of chromosome partition is discussed in this review. In addition , findings on partition mechanisms of plasmids are described. ANALYSIS OF GENES INVOLVED IN CHROMOSOME PARTITION Two Categories of Mechanisms
Two categories of mechanisms are involved in the overall process from termination of chromosome replication to partition of the daughter chromo somes into daughter cells upon cell division. The first category includes decatenation of chromosome catenanes, resolution of chromosome dimers or oligomers, and other topological events preparatory to the separation of daughter chromosomes (for a review, see 19). The second category consists of mechanisms for the active positioning of daughter chromosomes at cell quarter sites (12). Bacterial par Mutants Defective in Topoisomerases
Conditionally lethal par mutants of E. coli are capable of DNA synthesis and septation for some time after transfer to a nonpermissive temperature, but have lost control ovcr the spatial location of the septa. During incubation at nonpermissive temperature, these mutants form elongated cells with a large conglomerated nucleoid or nucleoids. This kind of abnormal cell formation has been called the "Par phenotype." In some cases, abnormal cell division occurs near the ends of elongated cells, producing anucleate cells. Some par mutants have been found to be defective in the genes coding for
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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topoisomerases. The parA mutant carries at least two temperature-sensiti ve mutations; one of them is probably located in the gyrE gene encoding the B subunit of DNA gyrase and may be responsible for the Par phenotype (20). The parD mutant carries an amber mutation in the gyrA gene encoding the A subunit of DNA gyrase, and the formation of elongated cells is probably due to the gyrA mutation rather than to another mutation present in the mutant and assigned to a separate gene called parD (21, 22). In the temperature-sensitive gyrE mutant, which is defective in the B subunit of DNA gyrase, a catenane of replicated daughter chromosomal DNA molecules is produced at nonper missive temperature (23, 24). Therefore, the daughter chromosomes cannot be physically separated.
The pare mutation is located at 6 5 min on the chromosome map (25). The nucleotide sequence of the parC gene was determined (26) . The pure gene encodes a 81.2-kDa protein, and the deduced amino acid sequence of the protein has homology to that of the A subunit of DNA gyrase. Another newly discovered gene, parE, which is located about 5 kilobases (kb) upstream from the pare gene and codes for a ca. 70-kDa protein, was sequenced, and the deduced amino acid sequence shows that the gene product (66.7 kDa) has homology to thc gyrase B subunit. Mutants of this gene show thc typical Par phenotype at nonpcrmissive temperature; thus thc gene was named parE. Relaxation activity of supercoiled plasmid molecules is enhanced in vitro in the combined crude cell lysates prepared from the Pare and ParE overpro ducers. The pare and parE genes code for the subunits of a newly discovered topoisomerase, named topoisomerase IV. The complex of Pare and ParE proteins causes a decrease of superhelicity as does eukaryotic type II topoisomerase (26), whereas bacterial DNA gyrase causes an increase of superhelicity. A tapA mutation causing a defect in topoisomerase I can be compensated in vivo by increasing the gene dosage of both pare and parE (26). Pare protein is specifically associated with the inner membrane, suggesting that topoisomerase IV is a membrane-bound enzyme (25; J. Kato and H. Ikeda, unpublished information). The parC and parE genes of Salmonella typhimurium encode homologues of the E. coli Pare and ParE proteins, respectively (27). The parE gene is about 5 kb upstream of the pare gene, and a third gene, parF, is just downstream of the pare gene. The DNA sequence of the S. typhimurium parF gene was determined and could encode a protein with a hydrophobic amino terminus (28). It was speculated that the ParF protein interacts with ParC and ParE to anchor these proteins to the membrane. Some other par mutants exhibit an inhibition of cell division, probably by the SOS response. For example, the parE mutant is an allele of the dnaG gene, which encodes a primase essential for DNA replication, and the Par phenotype observed at nonpermissive temperature reflects the inhibition of
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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HIRAGA
cell division associated with perturbed DNA synthesis (29, 30). The pcsA mutant (31), which shows a cold-induced Par phenotype, induces the RecA protein at nonpermissive temperature, suggesting that the Par phenotype of the pcsA mutant also reflects the inhibition of cell division by SOS-associated division inhibitors. Therefore these mutations causing SOS-associated cell division inhibition do not appear to belong to the category of mutations that specifically affect partition mechanisms. The dif(deletion-induced filamentation) site, which lies within the terminal region of the E. coli chromosome, may act to resolve dimeric chromosomes produced by sister chromatid exchange. The dif site is a cis-acting, recA independent recombination site. Deletion of the dif site causes the Dif phe notype, characterized by formation of a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system (32 see also 33, 34). This finding shows that site-specific resolution of chromosome dimers is also required for chromosome partition. Three new classes of cell cycle mutants showing increased ploidy were identified by selecting for cells that grow in the presence of camphor vapors (35, 36). The mutants, named mbr (moth ball resistant), map to four loci on the E. coli chromosome: mbrA maps to 68 min, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD, which may be allelic to rpoB, to 90 min. Based on several tests, mbrA may define a novel checkpoint that couples DNA replica tion to cell division. The mbrB mutant appears to be defective in the syn chrony of initiation of DNA replication. The mbrC and mbrD mutants are most likely defective in chromosome partitioning, although in precisely what way remains to be determined. We have isolated mutants characterized by the production, in rich media at 37°C, of a subpopulation of filamentous cells with abnormal nucleoids distributed irregularly along the cell. One of these mutants was demonstrated to have a defect in the ruvB gene located at 41 min on the E. coli chromosome (J. Peng, K. Yamanaka, S. Hiraga, unpublished results). The ruvB and ruvA genes constitute an operon, which belongs to the SOS regulon (37). The gene products are involved in DNA repair and recombination. Purified RuvB protein shows weak ATPase activity and binds ADP more strongly than ATP (37). The SOS repair system may be spontaneously induced in rich media in our mutant. Thus it appears that the primary defect in mutants of this type is not in chromosome partition.
muk Mutants: Mutants that Produce Chromosome-less Cells If the positioning of daughter chromosomes at the cell quarter positions is controlled by gene products, mutants defective in the relevant genes would be expected generally to be nonlethal. Cell division in these mutants would occur normally, and replicated daughter chromosomes would be appropriately de-
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
E. COLI
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catenated and folded. However, the daughter chromosomes would tend to be located close together in the position where the chromosome had been repli cated, because of the defect in the active positioning of daughter chromo somes at cell quarter positions. Therefore , one daughter cell carrying two copies of the chromosome and one daughter cell carrying no chromosome would be spontaneously produced by cell division in the growing cell popula tion at a non-negligible frequency as shown in Figure 1 (16; for a review, see 13). In a daughter cell that received two copies of the chromosome, the initiation of chromosome replication should be inhibited for one generation until the next cell division. We have developed techniques to isolate mutants that produce anucleate cells during cell division, and have indeed isolated mutants defective in chromosome positioning (16). One such isolation procedure makes use of a lacZ-bearing plasmid that is restrained from runaway replication and from full lacZ expression by copy control and lael repressor genes situated on the bacterial chromosome. Mutants that produce anucleate cells form blue colon ies on agar plates containing X-gal, because anucleate cells lack both res traints on {3-galactosidase synthesis from the lacZ genes. Mutants so obtained we designate muk (from the Japanese mukaku, meaning "anuc1eate") .
Wild
type
mukmutant
Newborn cell Imtiation of repl!cation Replication
(
.. ) ...
(I) t � t � t
•• )
Termination
...
•• ) I '"
Positiomng
Newborn cell Imtiation of repl!cation Replication
Termination No positiomng
� f
of
� (
f .. •• X."",--:;;i • ..-�) " '" J \
�
'-----y----'
Figure 1 Chromosome partitiuning in the E. coli wild-type strain and in a muk mutant defective in "positioning" of daughter chromosomes at cell quarter sites ( 1 6).
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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HIRAGA
Among these muk mutants, mukA1, located at 66 min on the E. coli chromosome, was selected for detailed analysis of its properties (16). This mutant shows all the expected properties of the hypothetical mutant described above. The mukA1 mutant produces normal-sized anucleate cells as about 3% of the population in an exponentially growing culture in a minimum-salt medium containing glucose. Fully replicated daughter chromosomes tend to be located close to each other. The intracellular location of the chromosome is broader in the mutant than in the parental wild-type strain. Abnormal pairs of one anucleate cell and one nucleate cell having two copies of chromosomal DNA can be observed at low frequency in a culture of the mutant. A wild-type chromosomal DNA segment that can complement the mukA1 mutation was cloned and sequenced (16, 38). Results obtained indicated that the mukA gene is identical to the tolC gene, which codes for an outer membrane protein with a signal peptide. The tolC mutants show pleiotropic properties, tolerance to colicin El protein, slow growth, hypersensitivity to sodium dodecyl sulfate and drugs, and abnormal expression of outer membrane proteins OmpF and OmpC (for a review, see 39). In addition, TolC protein is specifically required for hemolysin secretion and might be a component of the secretion apparatus allowing a specific interaction between the inner and outer mem branes (40) . The molecular mechanism of the TolC (MukA) protein in chromosome positioning is still unknown. A mutation belonging to another linkage group , mukB106, is located at 21 min on the E. coli chromosome (17) . The mukB gene has been found to code for a large protein that has a surprising predicted secondary structure and interesting properties in vitro as described below. THE mukB GENE AND ITS PRODUCT Properties of the mukB Mutants
The original mukB106 mutant grows nearly normally but spontaneously produces anucleate cells of normal size (about 5% of the population) during growth at 22 °e. The mutant shows temperature-sensitive growth and forms minute colonies at 42 °e in rich medium. A mukB-disrupted null mutant is very temperature sensitive and cannot form colonies at 42 °e in rich medium. The null mutant grows nearly normally at 22°C, but spontaneously produces anucleate cells of normal cell size. In addition to anucleate cells there are pairs of cells: one that is anucleate with about 1 unit of cell mass and one that is nucleate of 1-2 units of cell mass (see Figure 1) . There is also another type of cell pair: one nucleate cell with about two copies of chromosomal DNA and one cell having small amounts (5-20%) of chromosomal DNA at the cell pole near the division site, suggesting a guillotine effect by septal closure on a chromosome. When these muk mutants grown at 22°C are transferred to
E. COLl
CHROMOSOME PARTITION
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Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
42 °e, nucleate cells elongate and nucleoids become irregularly distributed along the elongated cells within 3-4 h (17). During prolonged incubation at 42 °e, the elongated cells gradually divide, resulting in normal-sized anu cleate cells and 40-60% as many normal-sized nucleate cells carrying large
quantities of DNA (18). These results suggest that the MukB protein is required for chromosome positioning at 22 °e through 42 °e. In the mukB mutants, cell division is partially inhibited and delayed at 42 °e. It has been demonstrated that the production of anucleate cells in the mukB mutants is n ot due to inhibition of DNA replication. Flow cytometric an alysi s of the mukB106 mutant showed that the control of initiation of chromosome replication is normal, and all replication origins within any individual cell initiate simultaneously, even in the population at intermediate temperature containing some elongated cells (17).
Predicted Secondary Structure of MukB Protein A wild-type E. coli DNA segment that complements the mukB106 mutation was cloned and sequenced (17). The mukB gene codes for a protein of 176,826 daltons consisting of 1543 amino acids. The gene is the largest described so far in E. coli. Computer analysis of the deduced amino acid sequence showed that the MukB protein is hydrophilic with no highly hydrophobic regions, such as are typical of the membrane domains of integral membrane proteins. MukB protein was predicted to have five domains showing distinguishable secondary structure (Figure 2). The amino-terminal domain I (amino acid residues 1-338) was predicted to be globular and to include a nucleotide binding consensus sequence. Domains II (residues 339-665) and IV (residues 934-1116) were predicted to be highly a-helical and to show an extended coiled-coil structure. These a-helical domains have seven-residue repeats, consistent with the ability to form a coiled-coil. The two a-helical domains were found to exhibit a heptapeptide repeat motif ab c d e f g; hydrophobic residues were enriched at positions a and d, and a periodicity of negatively charged residues (Asp and Glu) and positively charged residues (Lys, Arg, and His) was observed in these a-helical regions. These results are consistent with an a-helical coiled-coil conformation (41-44) in these regions. Domain III (residues 666-933) was predicted to be globular. The carboxyl-terminal region of domain V (residues 1116-1534) was also predicted to be globular. A subregion (residues 1364-1525) of domain V is rich in cysteine and the positively charged residues arginine and lysine. Within the carboxyl-terminal region of domain V are three putative "zinc finger"-like structures. Loops may be formed around the central atom of Zn (or another metal) . Four cysteine residues provide the linkers between consecutive loops in structures I and II. Three cysteine residues and one histidine residue provide the linkers in
292
HIRAGA
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
Molecular weight
177
kDa ( 1534 amino acids)
Secondary structure helix I
111111_11111111 1 101111111111111111111 II p sheet IIUII lID I I II I n UIII " II1111 IIIIIU I a
pturn
,IIIIII1I1J1mlll ,II ,111,�IIJII,OOI,IIII,IIIIIIIIIIIIIIIIIIIIII� 1
Domain
500
I
N
II
1000
ill
1600
N
V
: •••tll .. 11m. ........... /' + +
Further
Quick links to online content
Annu. Rev. Biochem. Copyright
© 1992
1992. 61:283-306
by Annual Reviews Inc. All rights reserved
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
CHROMOSOME AND PLASMID PARTITION IN ESCHERICHIA COLI Sofa Hiraga Department of Molecular Genetics, Institute for Medical Genetics, Kumamoto University Medical School, Kumamoto 862, Japan KEY WORDS:
chromosome partition, plasmid partition, MukB protein, ATPase. filamentous
protein polymer
CONTENTS INTRODUCTION.....................................................................................
283
ANALYSIS OF GENES INVOLVED IN CHROMOSOME PARTITION ..... ...........
286 286 286
Two Categories of Mechanisms. . . ... . . . .. . . . . ........... ....... . . . . ... . . . . . . . . . . . . . ..... . . . . . . Bacterial par Mutants Defective in Topoisomerases .... muk Mutants; Mutants that Produce Chromosome-less Cells......... ........ ........... THE mukB GENE AND ITS PRODUCT........................................................ Properties of the mukB Mutants......................... .............................. ........
Predicted Secondary Structure of MukB Protein....... .. .......... ..... . . . . . . . ........ . . . . Properties of MukB Protein in vitro. ...... . . .... . . . . . .... ........... . . . .. . . .. . . . . . . . . . . . .. . . . Filamentous Protein Polymers in E. coli.................................................... PARTITION MECHANISM OF PLASMlDS . ... ...... ............................... .......... Plasmid-Encoded Proteins and the cis-Acting Region of F and PI
Plasmids Essential for Partition..................................................
Partition Genes of the lncFfl Plasmids Rl and NRI ................. .... . . .... . . . . . . . . ... The cis-Acting par Site of pSCIOI Plasmid.............. ....................
Partition of a Mini-F Plasmid in a mukB-Disrupted Null Mutant......................
CONCLUDING REMARKS................................ . ... ... . .... ... . ..................
288 290 290 291
293
294 295 295
301
302 302 303
INTRODUCTION In bacteria, chromosomal DNA is replicated and daughter chromosome mole cules are accurately partitioned into daughter cells prior to cell division. 0066-4154/92/0701-0283$02.00
283
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
284
HIRAGA
Daughter cells almost always receive at least one copy of the chromosome; anucleate (chromosome-less) cells account for less than 0.03 % of the popula tion in growing cultures of wild-type strains of Escherichia coli. Before septation at midcell, daughter chromosome molecules are separated from each other and located at the cell quarter positions (1/4 and 3/4 cell lengths), where the daughter chromosomes appear more compact than replicating chromo somes. Jacob et al (1) proposed a model in which the partitioning of replicated chromosomes and molecules of F plasmid (the classical E. coli sex factor) into daughter cells involves a physical connection between a daughter DNA strand and part of the cell envelope, and in which the driving force for segregation of the two daughter chromosomes and of F plasmids must be provided by insertion of new cell envelope material at midcell, i.e. between two attachment sites. A ccording to one version of this model, the chromo some replication origin oriC or its flanking region is the specific site that is attached to the cell envelope, and daughter chromosomes move to opposite directions by elongation of the envelope, in concert with the progress of chromosome replication. However, experimental results showed that growth of the cell wall takes place by random insertion over the entire surface during most of the E. coli cell cycle, although during septum formation peptidogly can precursors are inserted preferentially at midcell (2-4). A lthough biochemical data showed temporary association of the bacterial membrane with the oriC regions of the bacterial chromosome and with oriC plasmids (minichromosomes) (for example, 5-7), it is doubtful that the membrane attachment of the oriC region actually plays an essential role in partition of the chromosome and of oriC plasmids to daughter cells in vivo. In the absence of selective pressure, oriC plasmids are unstably maintained in a population of actively dividing bacterial cells, and are lost rapidly from the bacterial population. They do not have a partition mechanism, but are parti tioned essentially at random into daughter cells. By contrast, an oriC plasmid earrying the sopA, sopB, and sopC genes (parA, parB, and parC) of the F plasmid is stably maintained in spite of the low copy number of plasmid molecules per cell (8). Therefore it is unlikely that oriC and/or its flanking regions play an essential role in chromosome partitioning. The two strands of the double-stranded chromosomal DNA in a newborn cell can be distinguished by the fact that one strand (the younger strand) was formed during the most recent round of replication and the other (the older strand) was formed during some earlier round of replication. Upon replica tion, the younger and older chromosomes separate from one another to eventually reside within the complementary new daughter cells. The direc tions in which the new chromosomes are partitioned can also be easily discriminated by the fact that every cell has a younger polar cap, formed at the
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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previous division, and an older polar cap formed during some earlier division. Does the cell distinguish between the two daughter chromosomes in conjunc tion with the direction of partition? To answer the question , studies have been made of the partitioning of younger and older daughter chromosomes into daughter cells of E. coli during division, and it has been shown that there is a preferental segregation of the older chromosome towards the cell with the older polar cap (for a review, see 9). Helmstetter & Leonard (10 ) propose that the cell envelope contains a large number of sites capable of binding to the chromosomal replication origin, oriC, that a polymerizing DNA strand be comes attached to one of the sites at initiation of a round of replication, and that the attachment sites are distributed throughout the actively growing cell envelope, i. e. lateral envelope and septum, but not in the existing cell poles. This asymmetric distribution of oriC attachment sites could account for the experimentally observed nonrandom segregation of the older daughter chromosome with cell strain and growth rate. The multisite attachment con cept could also account for at least some of the instability with which minichromosomes are maintained (9). It has been found that the "positioning" of daughter chromosome molecules at the cell quarter pos itio ns req uir es po stre pli ca tion al protein synthesis (1 1 , 1 2; for a review, see 1 3 ). When protein synthesis is inhibited by starvation for amino acids or by the addition of an inhibitor of protein synthesis, a replicat ing chromosome completes its replication and the r esulting daughter chromo somes remain at the midcell, close to each other. When protein synthesis resumes by the addition of the amino acids or by removal of the inhibitor, the daughter chromosomes can rapidly move from midcell to the cell quarter positions before a detectable increase in cell lcngth occurs (1 2). This suggests that daughter chromosomes are transported by an unknown mechanism, but not by elongation of the cell envelope itself. The positioning of daughter chromosomes from midcell to cell quarter positions after resumption of protein synthesis is achieved even in the presence of an inhibitor of DNA gyrase, nalidixic acid (50 JLg/ml), or novobiocin (l00 JLg/ml). Moreover, it was found that the positioning after resumption of protein synthesis is achiev ed even when the initiation of chromosome replication is inhibit ed using a temperature-sensitive dnaC mutant, which is defective in initiation of chromosome replication at a nonpermissive temperature. Thus the reinitiation of chromosome r eplic atio n is not required for the positioning of dau ghter chromosomes. The results described above strongly indicate that E. coli has a mechanism, albeit unknown, for positioning daughter chromosomes. This mechanism may be promoted by events that are dependent on pos trep lic ation al protein synthesis (1 2). The events may also promote septum formation, causing the cell to enter the D (division) period from the C period (for a review, see 1 4). Individual chromosomes are fixed in position within growing ,
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
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cells both before and during replication, but they are rapidly moved apart by a fixed distance (unit length) immediately after replication has been completed (15). Understanding how daughter chromosomes are positioned at cell quarter positions before cell division will provide insights into the fundamental process of partition. To analyze the molecular mechanism of chromosome positioning, isolation and characterization of mutants defective in this mech anism are advantageous. Techniques have been developed by us to isolate mutants that produce, upon cell division and at a non-negligible frequency, one anucleate daughter cell of normal cell sizc and one nucleate daughter cell carrying two copies of daughter chromosomes (16). Many mutants have been isolated by using these techniques. Our recent results indicate that a newly discovered gene, mukB, is involved in chromosome positioning at the cell quarter positions. The amino acid sequence deduced from the nucleotide sequence of the cloned mukB gene and the properties of the purified MukB protein suggest that the MukB protein is the first candidate found for a force-generating (mechanochemical) enzyme in bacteria (17, 18). The mech anism of chromosome partition is discussed in this review. In addition , findings on partition mechanisms of plasmids are described. ANALYSIS OF GENES INVOLVED IN CHROMOSOME PARTITION Two Categories of Mechanisms
Two categories of mechanisms are involved in the overall process from termination of chromosome replication to partition of the daughter chromo somes into daughter cells upon cell division. The first category includes decatenation of chromosome catenanes, resolution of chromosome dimers or oligomers, and other topological events preparatory to the separation of daughter chromosomes (for a review, see 19). The second category consists of mechanisms for the active positioning of daughter chromosomes at cell quarter sites (12). Bacterial par Mutants Defective in Topoisomerases
Conditionally lethal par mutants of E. coli are capable of DNA synthesis and septation for some time after transfer to a nonpermissive temperature, but have lost control ovcr the spatial location of the septa. During incubation at nonpermissive temperature, these mutants form elongated cells with a large conglomerated nucleoid or nucleoids. This kind of abnormal cell formation has been called the "Par phenotype." In some cases, abnormal cell division occurs near the ends of elongated cells, producing anucleate cells. Some par mutants have been found to be defective in the genes coding for
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topoisomerases. The parA mutant carries at least two temperature-sensiti ve mutations; one of them is probably located in the gyrE gene encoding the B subunit of DNA gyrase and may be responsible for the Par phenotype (20). The parD mutant carries an amber mutation in the gyrA gene encoding the A subunit of DNA gyrase, and the formation of elongated cells is probably due to the gyrA mutation rather than to another mutation present in the mutant and assigned to a separate gene called parD (21, 22). In the temperature-sensitive gyrE mutant, which is defective in the B subunit of DNA gyrase, a catenane of replicated daughter chromosomal DNA molecules is produced at nonper missive temperature (23, 24). Therefore, the daughter chromosomes cannot be physically separated.
The pare mutation is located at 6 5 min on the chromosome map (25). The nucleotide sequence of the parC gene was determined (26) . The pure gene encodes a 81.2-kDa protein, and the deduced amino acid sequence of the protein has homology to that of the A subunit of DNA gyrase. Another newly discovered gene, parE, which is located about 5 kilobases (kb) upstream from the pare gene and codes for a ca. 70-kDa protein, was sequenced, and the deduced amino acid sequence shows that the gene product (66.7 kDa) has homology to thc gyrase B subunit. Mutants of this gene show thc typical Par phenotype at nonpcrmissive temperature; thus thc gene was named parE. Relaxation activity of supercoiled plasmid molecules is enhanced in vitro in the combined crude cell lysates prepared from the Pare and ParE overpro ducers. The pare and parE genes code for the subunits of a newly discovered topoisomerase, named topoisomerase IV. The complex of Pare and ParE proteins causes a decrease of superhelicity as does eukaryotic type II topoisomerase (26), whereas bacterial DNA gyrase causes an increase of superhelicity. A tapA mutation causing a defect in topoisomerase I can be compensated in vivo by increasing the gene dosage of both pare and parE (26). Pare protein is specifically associated with the inner membrane, suggesting that topoisomerase IV is a membrane-bound enzyme (25; J. Kato and H. Ikeda, unpublished information). The parC and parE genes of Salmonella typhimurium encode homologues of the E. coli Pare and ParE proteins, respectively (27). The parE gene is about 5 kb upstream of the pare gene, and a third gene, parF, is just downstream of the pare gene. The DNA sequence of the S. typhimurium parF gene was determined and could encode a protein with a hydrophobic amino terminus (28). It was speculated that the ParF protein interacts with ParC and ParE to anchor these proteins to the membrane. Some other par mutants exhibit an inhibition of cell division, probably by the SOS response. For example, the parE mutant is an allele of the dnaG gene, which encodes a primase essential for DNA replication, and the Par phenotype observed at nonpermissive temperature reflects the inhibition of
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cell division associated with perturbed DNA synthesis (29, 30). The pcsA mutant (31), which shows a cold-induced Par phenotype, induces the RecA protein at nonpermissive temperature, suggesting that the Par phenotype of the pcsA mutant also reflects the inhibition of cell division by SOS-associated division inhibitors. Therefore these mutations causing SOS-associated cell division inhibition do not appear to belong to the category of mutations that specifically affect partition mechanisms. The dif(deletion-induced filamentation) site, which lies within the terminal region of the E. coli chromosome, may act to resolve dimeric chromosomes produced by sister chromatid exchange. The dif site is a cis-acting, recA independent recombination site. Deletion of the dif site causes the Dif phe notype, characterized by formation of a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system (32 see also 33, 34). This finding shows that site-specific resolution of chromosome dimers is also required for chromosome partition. Three new classes of cell cycle mutants showing increased ploidy were identified by selecting for cells that grow in the presence of camphor vapors (35, 36). The mutants, named mbr (moth ball resistant), map to four loci on the E. coli chromosome: mbrA maps to 68 min, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD, which may be allelic to rpoB, to 90 min. Based on several tests, mbrA may define a novel checkpoint that couples DNA replica tion to cell division. The mbrB mutant appears to be defective in the syn chrony of initiation of DNA replication. The mbrC and mbrD mutants are most likely defective in chromosome partitioning, although in precisely what way remains to be determined. We have isolated mutants characterized by the production, in rich media at 37°C, of a subpopulation of filamentous cells with abnormal nucleoids distributed irregularly along the cell. One of these mutants was demonstrated to have a defect in the ruvB gene located at 41 min on the E. coli chromosome (J. Peng, K. Yamanaka, S. Hiraga, unpublished results). The ruvB and ruvA genes constitute an operon, which belongs to the SOS regulon (37). The gene products are involved in DNA repair and recombination. Purified RuvB protein shows weak ATPase activity and binds ADP more strongly than ATP (37). The SOS repair system may be spontaneously induced in rich media in our mutant. Thus it appears that the primary defect in mutants of this type is not in chromosome partition.
muk Mutants: Mutants that Produce Chromosome-less Cells If the positioning of daughter chromosomes at the cell quarter positions is controlled by gene products, mutants defective in the relevant genes would be expected generally to be nonlethal. Cell division in these mutants would occur normally, and replicated daughter chromosomes would be appropriately de-
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catenated and folded. However, the daughter chromosomes would tend to be located close together in the position where the chromosome had been repli cated, because of the defect in the active positioning of daughter chromo somes at cell quarter positions. Therefore , one daughter cell carrying two copies of the chromosome and one daughter cell carrying no chromosome would be spontaneously produced by cell division in the growing cell popula tion at a non-negligible frequency as shown in Figure 1 (16; for a review, see 13). In a daughter cell that received two copies of the chromosome, the initiation of chromosome replication should be inhibited for one generation until the next cell division. We have developed techniques to isolate mutants that produce anucleate cells during cell division, and have indeed isolated mutants defective in chromosome positioning (16). One such isolation procedure makes use of a lacZ-bearing plasmid that is restrained from runaway replication and from full lacZ expression by copy control and lael repressor genes situated on the bacterial chromosome. Mutants that produce anucleate cells form blue colon ies on agar plates containing X-gal, because anucleate cells lack both res traints on {3-galactosidase synthesis from the lacZ genes. Mutants so obtained we designate muk (from the Japanese mukaku, meaning "anuc1eate") .
Wild
type
mukmutant
Newborn cell Imtiation of repl!cation Replication
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of
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f .. •• X."",--:;;i • ..-�) " '" J \
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Figure 1 Chromosome partitiuning in the E. coli wild-type strain and in a muk mutant defective in "positioning" of daughter chromosomes at cell quarter sites ( 1 6).
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Among these muk mutants, mukA1, located at 66 min on the E. coli chromosome, was selected for detailed analysis of its properties (16). This mutant shows all the expected properties of the hypothetical mutant described above. The mukA1 mutant produces normal-sized anucleate cells as about 3% of the population in an exponentially growing culture in a minimum-salt medium containing glucose. Fully replicated daughter chromosomes tend to be located close to each other. The intracellular location of the chromosome is broader in the mutant than in the parental wild-type strain. Abnormal pairs of one anucleate cell and one nucleate cell having two copies of chromosomal DNA can be observed at low frequency in a culture of the mutant. A wild-type chromosomal DNA segment that can complement the mukA1 mutation was cloned and sequenced (16, 38). Results obtained indicated that the mukA gene is identical to the tolC gene, which codes for an outer membrane protein with a signal peptide. The tolC mutants show pleiotropic properties, tolerance to colicin El protein, slow growth, hypersensitivity to sodium dodecyl sulfate and drugs, and abnormal expression of outer membrane proteins OmpF and OmpC (for a review, see 39). In addition, TolC protein is specifically required for hemolysin secretion and might be a component of the secretion apparatus allowing a specific interaction between the inner and outer mem branes (40) . The molecular mechanism of the TolC (MukA) protein in chromosome positioning is still unknown. A mutation belonging to another linkage group , mukB106, is located at 21 min on the E. coli chromosome (17) . The mukB gene has been found to code for a large protein that has a surprising predicted secondary structure and interesting properties in vitro as described below. THE mukB GENE AND ITS PRODUCT Properties of the mukB Mutants
The original mukB106 mutant grows nearly normally but spontaneously produces anucleate cells of normal size (about 5% of the population) during growth at 22 °e. The mutant shows temperature-sensitive growth and forms minute colonies at 42 °e in rich medium. A mukB-disrupted null mutant is very temperature sensitive and cannot form colonies at 42 °e in rich medium. The null mutant grows nearly normally at 22°C, but spontaneously produces anucleate cells of normal cell size. In addition to anucleate cells there are pairs of cells: one that is anucleate with about 1 unit of cell mass and one that is nucleate of 1-2 units of cell mass (see Figure 1) . There is also another type of cell pair: one nucleate cell with about two copies of chromosomal DNA and one cell having small amounts (5-20%) of chromosomal DNA at the cell pole near the division site, suggesting a guillotine effect by septal closure on a chromosome. When these muk mutants grown at 22°C are transferred to
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42 °e, nucleate cells elongate and nucleoids become irregularly distributed along the elongated cells within 3-4 h (17). During prolonged incubation at 42 °e, the elongated cells gradually divide, resulting in normal-sized anu cleate cells and 40-60% as many normal-sized nucleate cells carrying large
quantities of DNA (18). These results suggest that the MukB protein is required for chromosome positioning at 22 °e through 42 °e. In the mukB mutants, cell division is partially inhibited and delayed at 42 °e. It has been demonstrated that the production of anucleate cells in the mukB mutants is n ot due to inhibition of DNA replication. Flow cytometric an alysi s of the mukB106 mutant showed that the control of initiation of chromosome replication is normal, and all replication origins within any individual cell initiate simultaneously, even in the population at intermediate temperature containing some elongated cells (17).
Predicted Secondary Structure of MukB Protein A wild-type E. coli DNA segment that complements the mukB106 mutation was cloned and sequenced (17). The mukB gene codes for a protein of 176,826 daltons consisting of 1543 amino acids. The gene is the largest described so far in E. coli. Computer analysis of the deduced amino acid sequence showed that the MukB protein is hydrophilic with no highly hydrophobic regions, such as are typical of the membrane domains of integral membrane proteins. MukB protein was predicted to have five domains showing distinguishable secondary structure (Figure 2). The amino-terminal domain I (amino acid residues 1-338) was predicted to be globular and to include a nucleotide binding consensus sequence. Domains II (residues 339-665) and IV (residues 934-1116) were predicted to be highly a-helical and to show an extended coiled-coil structure. These a-helical domains have seven-residue repeats, consistent with the ability to form a coiled-coil. The two a-helical domains were found to exhibit a heptapeptide repeat motif ab c d e f g; hydrophobic residues were enriched at positions a and d, and a periodicity of negatively charged residues (Asp and Glu) and positively charged residues (Lys, Arg, and His) was observed in these a-helical regions. These results are consistent with an a-helical coiled-coil conformation (41-44) in these regions. Domain III (residues 666-933) was predicted to be globular. The carboxyl-terminal region of domain V (residues 1116-1534) was also predicted to be globular. A subregion (residues 1364-1525) of domain V is rich in cysteine and the positively charged residues arginine and lysine. Within the carboxyl-terminal region of domain V are three putative "zinc finger"-like structures. Loops may be formed around the central atom of Zn (or another metal) . Four cysteine residues provide the linkers between consecutive loops in structures I and II. Three cysteine residues and one histidine residue provide the linkers in
292
HIRAGA
Annu. Rev. Biochem. 1992.61:283-306. Downloaded from www.annualreviews.org by University of Wyoming on 09/30/13. For personal use only.
Molecular weight
177
kDa ( 1534 amino acids)
Secondary structure helix I
111111_11111111 1 101111111111111111111 II p sheet IIUII lID I I II I n UIII " II1111 IIIIIU I a
pturn
,IIIIII1I1J1mlll ,II ,111,�IIJII,OOI,IIII,IIIIIIIIIIIIIIIIIIIIII� 1
Domain
500
I
N
II
1000
ill
1600
N
V
: •••tll .. 11m. ........... /' + +
