dna transport in bacteria - Nature

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DNA TRANSPORT IN BACTERIA Jeff Errington, Jonathan Bath and Ling Juan Wu DNA transport is important in various biological contexts — particularly chromosome segregation and intercellular gene transfer. Recently, progress has been made in understanding the function of a family of bacterial proteins involved in DNA transfer, and we focus here on one of the best-understood members, SpoIIIE. Studies of SpoIIIE-like proteins show that they might couple DNA transport to processes such as cell division, conjugation (mating) and the resolution of chromosome dimers. MOBILE GENETIC ELEMENTS

Plasmids, transposons and insertion elements that can readily spread from genome to genome and/or from cell to cell. ENDOSPORE

A type of spore that develops within the cytoplasm of an enveloping mother cell or sporangium. EUBACTERIA

All prokaryotes excluding the Archaea. NUCLEOID

Equivalent to the nucleus of bacteria, in the absence of a nuclear membrane.

The movement of DNA within and between bacterial cells is required for fundamental processes such as the replication and segregation of chromosomes during cellcycle progression, the packaging of DNA into bacteriophage, and the transfer of MOBILE GENETIC ELEMENTS. Some bacteria have life cycles involving complex morphological changes that require chromosomal DNA to be translocated. An indication of the diversity of processes is provided in BOX 1. In general, relatively little is known about the mechanisms that underlie these processes, but it is emerging that, at least in some cases, motor proteins act directly on DNA to bring about movement. In this review, we summarize recent developments in understanding the functions of a group of proteins that mediate DNA transfer during chromosome segregation and conjugation (mating), and indicate how this field is likely to develop during the next few years. The SpoIIIE DNA transporter

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Correspondence to J.E. e-mail: [email protected]

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In bacteria, a protein called SpoIIIE was first identified through its role in chromosome segregation in a specialized form of cell division that occurs during ENDOSPORE differentiation in Bacillus subtilis. This rodshaped organism normally divides medially to produce two similar daughter cells. However, during spore formation, the division site is moved close to one end of the rod to generate a small prespore and a much bigger mother cell (FIG. 1). spoIIIE mutant cells have a striking phenotype in which only a small part of the circular chromosome is segregated into the prespore compartment1. The SpoIIIE protein is required for the transport of the prespore chromosome into the small polar compartment after the septum that separates off the

two cells has been formed2. The amino-terminal domain acts as a membrane anchor that also targets the SpoIIIE protein to the constricting division septum, and the carboxy-terminal domain of the protein has a DNA-tracking activity that could, in principle, act directly on the DNA to transport it through a pore in the septum3,4 (FIG. 1). Although the function of SpoIIIE in sporulating cells seems to be a specialized solution to the problem of segregating DNA during asymmetric cell division, the widespread conservation of the spoIIIE gene, almost throughout the EUBACTERIA, indicated that it might have a more general function. One early clue in support of a more general function came from the unexpected finding that expression of the spoIIIE gene is constitutive and not restricted to sporulation5. More recently, it was shown6 that SpoIIIE is required for the completion of chromosome segregation in vegetative cells of B. subtilis that have been perturbed such that the division septum has an increased likelihood of closing around the DNA. (Normally, the replicated sister chromosomes will have separated and cleared the mid-cell region well before the division septum forms.) Furthermore, spoIIIE mutations have a synthetic-lethal phenotype when combined with a mutation, smc7, which affects NUCLEOID structure and chromosome segregation. Thus, one general function of SpoIIIE seems to be in clearing DNA from the site of cell division if the normal close coordination of chromosome segregation and cell division fails. However, work on related proteins in other organisms has raised the possibility that this large protein might have several functions associated with the late stages of cell division in a broad range of bacteria.

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Box 1 | Processes involving intercellular DNA transport

a | Many bacteria are naturally competent for DNA transformation,

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which involves direct uptake and incorporation into their genome of DNA from the environment. In all of the well-studied bacterial transformation systems (including Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae, and Acinetobacter calcoaceticus) it has been shown that ssDNA is transported into the cytoplasm, with the non-transported strand being degraded before or during transport. Although Gram-positive and Gram-negative bacteria differ in various cell surface properties, many of the characterized proteins involved in DNA uptake are conserved in the two systems, and it is believed that the mechanism of DNA transport across the cytoplasmic membrane is conserved48. Infection b | Bacterial viruses (bacteriophages) also need to transport their (passive) genomes into cells. Although a range of strategies are used for DNA b transport during infection, the rate of transport is generally much higher than for transformation or other mechanisms, and it probably occurs through facilitated diffusion. During infection, phage DNA is translocated through a channel or pore consisting of both phage and host proteins, or only phage proteins49. At the end of Packaging the infectious cycle, another DNA transfer process is needed, to (ATP dependent) package the newly replicated phage genomes into the preformed c procapsids. This process requires energy, and it is unique in that the barrier crossed by the DNA molecule is not a membrane but the pore of a transport protein called the ‘portal protein’ or ‘head–tail connector’50. c | Conjugation is a third mechanism of intercellular DNA transfer in bacteria. Transfer occurs in a polarized manner, generally from a donor cell bearing a conjugative plasmid or transposable element to a plasmid-free recipient. During conjugation, DNA must be passed through the envelopes of both the donor and the recipient cells. Conjugative transfer of DNA in Gram-negative and most Grampositive bacteria occurs essentially by the same mechanism, involving many plasmid-encoded proteins, and only one linear d strand of DNA is transferred from the donor to the recipient cell. However, in the Gram-positive soil bacterium Streptomyces a different mechanism is probably used because a single plasmidencoded protein, Tra, is sufficient to promote plasmid transfer and the movement of chromosome genes between mating cells. d | In some bacteria with specialized life cycles (for instance, Hyphomonas spp.), new cells are formed by budding from the distal end of a stalk or prosthecum. For the newborn cell to acquire a genome, the DNA must be passed through the prosthecum, implying the intervention of some kind of DNA transportation machinery51.

FtsK: coupling DNA transport to cell division

The ftsK gene of Escherichia coli was originally discovered on the basis of a mutation that conferred a temperature-sensitive defect in cell division8, and later by a mutation that affected survival in stationary phase9. Surprisingly, sequence analysis revealed that this gene is a clear homologue of spoIIIE, and it is now clear that most bacteria have at least one member of this gene family (FIG. 2). The original ftsK mutation affected the amino-terminal coding region of the protein (again the domain required for targeting the protein to the celldivision septum10,11). The effect of this mutation on division was at a late step, just before cell separation, distinct from previously described cell-division mutations8. So far, the basis for the effect on cell division has not been worked out, and there is no equivalent phenotype in B. subtilis spoIIIE mutants (A. L. Marston and

L.J.W., unpublished data). Interestingly, however, mutation of spoIIIE can produce a phenotype in which an unusual membrane fusion event that occurs during sporulation is blocked12. About one hour after asymmetric cell division, the small prespore compartment is engulfed by the mother cell in a phagocytosis-like process13,14. This process culminates with fusion of the enveloping mother cell membranes at the distal end of the prespore. The prespore then completes its differentiation into a spore, completely enclosed within the cytoplasm of the mother cell. In spoIIIE mutants, the final step of membrane fusion fails, suggesting that SpoIIIE somehow catalyses this event12. It follows that the late-division phenotype of E. coli ftsK mutants could also be associated with a membrane fusion event late in closure of the division septum. Many of the chromosomally encoded SpoIIIE homologues

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Figure 1 | Sporulation and the role of SpoIIIE. a | Upper panels show a schematic representation of chromosome segregation in sporulating cells of Bacillus subtilis. The asymmetrically positioned septum closes around one of the chromosomes (blue), initially trapping only a minor part of this chromosome in the small prespore compartment. The larger part of the chromosome is then progressively transferred through the septum to complete the segregation process. Lower panels: examples of images of cells at successive stages of transfer. These cells have been double stained with a membrane dye, FM5-95 (purple), showing the cell outline and asymmetric septum, and a DNA dye, DAPI (blue), showing the progressive transfer of the chromosome. b | The upper panel shows a schematic of an intermediate in prespore chromosome segregation, with the SpoIIIE protein indicated as a red ring around the DNA at the leading edge of the septum. Below is an image of a sporulating cell of a spoIIIE mutant doubly stained for chromosomal DNA (DAPI, blue) and for SpoIIIE protein (by immunofluorescence using antibodies against SpoIIIE, red3). The spoIIIE gene has a point mutation that prevents DNA transfer but not protein localization. The protein is clearly located at the centre of the asymmetric septum, corresponding to the position where the prespore chromosome is bisected. c | Enlarged schematic of the closing asymmetric septum, showing SpoIIIE anchored at the leading edge of the closing septum through its four transmembrane segments, and with its carboxy-terminal transfer domain interacting directly with the chromosomal DNA within the septal annulus.

HOLLIDAY JUNCTION INTERMEDIATE

An X-shaped DNA structure formed during recombination, after strand exchange between homologous DNA duplexes. WALKER A AND B MOTIFS

Highly conserved shortsequence motifs found in numerous proteins that bind and sometimes hydrolyse ATP.

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seem to have an unusual amino-terminal membrane anchor, composed of four transmembrane segments, in which the fourth segment is unusually long and is preceded by a relatively conserved motif that is rich in glycines and hydrophobic residues (FIG. 2). It seems possible that this special topology is associated with targeting the proteins to the curved membrane at the leading edge of the septum or facilitating various membrane fusion events. Indeed, an allele of spoIIIE in which septal targeting was abolished produces a substitution of the completely conserved glycine in the conserved motif 3. E. coli FtsK is considerably larger than most other SpoIIIE-like proteins, mainly due to the presence of a long linker region between the membrane anchor and the carboxy-terminal transfer (Tra) domain. Alignment of many SpoIIIE homologues reveals that all of these proteins contain a hypervariable region in the position of the FtsK linker domain. This domain is generally rich in proline, glutamine and glutamate residues, and its length varies from about 100 amino acids in B. subtilis SpoIIIE to 850 residues in E. coli FtsK8. The function of this domain is not known, although its characteristics indicate that it might act as a flexible or elastic linker8. Because the space in the constricting septal annulus is potentially crowded, with a range of abundant proteins participating in the mechanics of cell division, the linker could act as a kind of spacer holding the carboxy-terminal Tra domain

away from the membrane and in the centre of the annulus where it can interact with the chromosome. Alternatively, the linker might be important for topological flexibility, and allow assembly of the active form of the protein — possibly a hexameric ring (see below). Mutation of the highly conserved putative Tra domain of E. coli ftsK does affect chromosome segregation15,16 but, surprisingly, the most prominent defect has now been shown to involve resolution of chromosome dimers rather than post-septational DNA transfer17–19 (FIG. 3). The circular nature of the chromosome in many bacteria imposes an intrinsic problem in that homologous recombination between sister chromosomes can result in formation of chromosome dimers. This is potentially catastrophic because a covalent dimer cannot be segregated at cell division. To overcome this problem, chromosome dimers are resolved by the Xer system. The system is composed of two recombinases, XerC and XerD, which catalyse recombination between two copies of the chromosomal site dif 20,21. We are tempted to speculate that FtsK, like SpoIIIE, can facilitate the movement of chromosomes, and that the role of FtsK in the Xer system is to bring the participating dif sites together. However, recent experiments indicate that although dif sites might meet in the absence of FtsK, the HOLLIDAY JUNCTION INTERMEDIATE that forms after sites have met cannot be processed19. Despite this result, we prefer not to rule out a role for FtsK in bringing dif sites together: at the least, FtsK could be required to bring together dif sites that happen to be trapped on opposite sides of a closing division septum (FIG. 3). Recently, it has emerged that B. subtilis has a dimerresolution system with homologues of dif, XerC (CodV) and XerD (RipX)22–25. However, this system seems less critical for survival of B. subtilis than it does for E. coli, and the possible role for SpoIIIE in B. subtilis is less clear-cut. At any rate, it seems that the SpoIIIE/FtsK family of chromosomal proteins might have various roles in processes associated with the completion of cell division in bacteria. Links to DNA transfer during mating

Another group of proteins showing weak similarity to the SpoIIIE/FtsK family is encoded by transmissible plasmids and transposons in a range of Gram-positive bacteria2,8. These proteins (generally called Tra proteins) seem to mediate transfer of DNA from the host cell containing the element — the ‘donor’ — to a recipient strain that is usually a plasmid- or transposon-free strain of a related organism. The sequence similarity spans a large portion of the carboxy-terminal transfer domain of SpoIIIE, including the prominent ATP WALKER A AND B MOTIFS and several other short motifs that are particularly conserved in the proteins encoded chromosomally (FIG. 2). In some cases, the plasmid proteins do not seem to have an amino-terminal hydrophobic domain (for example, Spi26), although at least two of the proteins are found in the membrane fraction27,28. The fact that the proteins with the strongest similarity to SpoIIIE are all encoded by plasmids of Gram-positive bacteria might simply reflect the evolutionary ori-




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Figure 2 | Domain structure of SpoIIIE and related proteins. In the motifs, almost completely conserved residues are shown in capitals and widely conserved residues in lower case. Hyphens indicate positions of low conservation and ‘x’ indicates a hydrophobic residue. Full alignments of SpoIIIE and related proteins can be obtained at the ProDom web site.

gins of the genes. By far the best-characterized conjugative DNA transfer system is that of the F factor from the Gram-negative bacterium E. coli K-12 (reviewed in REF. 29). In this, and several other related systems, the DNA seems to be transferred in a single-stranded (ssDNA) form through a process that involves many proteins. Several proteins are involved in mating pair formation (Mpf); in other words, bringing donor (plasmid-containing) and recipient (plasmid-free) cells together. Often this involves a specialized cell-surface appendage, the ‘sex pilus’. Other proteins are involved in the formation of a channel from donor to recipient. At the DNA level (reviewed in REF. 30), the donor DNA has a specific, cis-acting sequence, oriT, to which a RELAXOSOME COMPLEX of proteins is bound. A site-specific nick is made — by

RELAXOSOME COMPLEX

Complex of a Tra protein bound to its DNA recognition site (oriT), at which a singlestranded nick occurs to initiate DNA transfer. ROLLING-CIRCLE DNA REPLICATION

Form of DNA replication on a circular template in which a single DNA strand is synthesized, continuously displacing the non-template strand. DNA POLYMERASE III HOLOENZYME

Multisubunit protein complex responsible for the bulk of chromosome replication in bacteria.

XerC/XerD

FtsK

dif

Figure 3 | FtsK and the resolution of chromosome dimers. Any odd number of recombination events between two circular chromosomes will result in a dimer in which the two chromosomes are joined, head-to-tail, in a single circular molecule. Proper segregation requires that the dimer is resolved into its component monomers. Dimer resolution is catalysed by the Xer site-specific recombination system in a reaction that proceeds through a Holliday-junction intermediate. The carboxy-terminal domain of FtsK is also required for dimer resolution17, and there is evidence to suggest that it acts to ensure that the Holliday junction is in an appropriate conformation to allow the reaction to proceed to completion19.

the TraI protein in the case of F plasmid — and at this site ROLLING-CIRCLE DNA REPLICATION is initiated to provide the single-stranded DNA (ssDNA) that is transferred to the recipient. TraI also has a helicase activity that facilitates strand separation. A widely conserved DNA-binding protein, TraD (also called TraG and TrwB in other systems) couples the relaxosome to the membrane-spanning DNA transfer channel complex. This family of proteins has weak sequence similarity to SpoIIIE, particularly in the region of the Walker motifs. As discussed below, it is possible that these proteins have a direct role in DNA transfer. In contrast to the complexity of the F-like conjugation systems, it is intriguing that the SpoIIIE-like conjugation proteins of Gram-positive bacteria often reside on small plasmids, with no sign of the multiplicity of factors described above and only one plasmid-encoded protein being essential for DNA transfer31–33. In particular, the nicking enzyme and helicase, which generate the ssDNA substrate for transfer, seem to be absent. We speculate that DNA transfer during conjugation mediated by the SpoIIIE-like proteins on small plasmids in Gram-positive bacteria occurs by a double-stranded DNA (dsDNA) mechanism. At present, there are no published data that directly address this issue. However, in the example of B. subtilis sporulation, a good case can be made for the transfer involving dsDNA. This is based on the observation that, under well-controlled conditions, the time at which chromosome replication in preparation for sporulation is completed34–36 is ~30 min before the prespore chromosome is transferred across the septum37. As DNA transfer by SpoIIIE — and, indeed, sporulation — can occur in the presence of an inhibitor of the DNA POLYMERASE III HOLOENZYME (HPUra)35, it seems that DNA replication is not needed to restore a dsDNA genome in the prespore, so the transfer probably does not occur through a ssDNA intermediate (it seems unlikely that two single strands are transferred and then re-annealed). Although the nature of DNA transfer during conjugation is only just beginning to be understood, this process is very important in nature. Transfer systems are prevalent, and they can be extremely promiscuous, with vast taxonomic barriers readily being crossed. Thus, the RP4 plasmid was shown to be capable of directing its own transfer from E. coli to yeast38. The Ti plasmids of Agrobacterium tumefaciens have long been harnessed by molecular biologists because of their ability to transfer Ti DNA to plant cells39, and a recent report40 showed that the same plasmids could transfer DNA even to mammalian cells. Such systems are likely to increase in importance in terms of their use as tools for genetic manipulation in the future. Biochemistry of DNA transport

Biochemical experiments have recently added weight to the idea that SpoIIIE is a DNA pump4. The cytoplasmic carboxy-terminal domain of SpoIIIE has a DNA-dependent ATPase activity, suggesting that the energy required for chromosome transfer derives from nucleotide hydrolysis. The next step was to test the ability of the


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Figure 4 | DNA tracking assay for SpoIIIE. The helical nature of DNA makes it likely that as a protein tracks along the long axis of DNA there is relative rotation of DNA and protein (in the case of RNA polymerase, rotation has recently been shown using a single molecule technique54; BOX 2). The frictional forces opposing rotation might be large enough to result in transient supercoiling either side of the protein (positive supercoiling ahead of the protein and negative supercoiling in its wake). The tracking assay used for SpoIIIE4 was based on detection of transient supercoiling. Escherichia coli topoisomerase I will remove negative supercoils but not positive supercoils. So the presence of both the tracking protein and E. coli topoisomerase I should lead to the accumulation of positive supercoils in a plasmid DNA. The presence of positively supercoiled product in reactions containing both SpoIIIE and E. coli topoisomerase I suggested that SpoIIIE does indeed track along DNA. Furthermore, the failure of a nonhydrolysable ATP analogue to substitute for ATP suggested that the movement of SpoIIIE is driven by ATP hydrolysis rather than by nucleotide binding.

carboxy-terminal domain of SpoIIIE to move relative to the long axis of DNA. It was anticipated that SpoIIIE follows the helical path of DNA as it tracks, and that the frictional forces impeding the relative rotation of protein and DNA might result in the generation of transient supercoiling. An assay based on detection of transient supercoiling (FIG. 4) was used to infer that the carboxy-terminal domain of SpoIIIE uses energy derived from ATP hydrolysis to drive movement of SpoIIIE relative to the long axis of the DNA to which it

binds4. The combination of an amino-terminal domain that anchors SpoIIIE to the septum, and a carboxy-terminal domain that couples ATP hydrolysis to movement relative to the long axis of DNA, gives SpoIIIE the potential to act as a DNA pump4. A more direct demonstration that SpoIIIE tracks along DNA awaits further study; perhaps the advent of single-molecule techniques could prove useful in this respect (BOX 2). The idea that SpoIIIE is a DNA pump raises several questions. The key question for those studying DNA motor proteins is the mechanism by which movement is coupled to the use of a nucleotide. It remains to be seen if a common mechanism will emerge. However, progress was made recently when the crystal structure of a putative conjugation protein, TrwB from plasmid R388 of E. coli, was solved41. The protein seems to form a hexameric ring, one face of which probably abuts the cytoplasmic membrane. The central channel of the hexamer is about 20-Å wide, increasing to about 22 Å at the end adjacent to the membrane. The cytoplasmic entrance of the channel, however, is plugged by a ring of asparagine residues and is restricted to ~8 Å in diameter. The authors suggested that the single DNA strand might pass through the central channel to be introduced into the transport complex formed by the Mpf proteins (see above). The protein probably uses energy from ATP hydrolysis to pump the ssDNA in the same way that ring helicases move along DNA42. It is proposed that ATP binding and hydrolysis triggers a molecular switch mechanism that affects the channel, which promotes DNA binding and translational movement. However, it is not yet clear how events such as the release of ssDNA from the relaxosome, the helicase activity of TraI, and rolling-circle replication are tied together. It is also not known how DNA passes through

Box 2 | Single molecule experiments Experiments with isolated Magnet single protein molecules provide a powerful complement to more traditional, relatively indirect studies of populations of molecules. In each of the experiments described below, a Rate b Stall force c Rotation the DNA motor protein under study is attached to a solid support and a bead of about 1 µm in diameter is attached to one end of a DNA molecule. Interaction between the motor protein and its substrate DNA serves to tether the bead to the solid support. a | If the motor protein tracks along DNA in the direction shown, the length of the tether will decrease and with it the freedom of the bead to move by Brownian motion52. This approach can be used to measure rates of movement and to obtain information about directionality. b | The bead can be captured with an optical trap53. This approach allows measurement of the stall force (the force required to prevent movement of the motor) and step size (the distance moved for each discrete step of the motor). c | Smaller fluorescent particles are attached to the bead, which in turn is attached to the DNA. Rotation of the DNA about its helical axis generated by movement of the motor protein is transmitted to the bead and can be detected by fluorescence microscopy54. This approach has been used to measure the rotation of DNA generated by a motor protein when attached to a solid support and could potentially be used to allow resolution of step sizes as small as a single base pair. These techniques, in combination with advances in the ability to detect single fluorophore molecules55, provide an experimental strategy for investigating the coupling between nucleotide consumption and movement.

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Figure 5 | Topological problems of chromosome–SpoIIIE interaction and the problem of directionality. We assume that SpoIIIE is a ring with a central cavity large enough to accommodate double-stranded DNA. If SpoIIIE were to accommodate both the clockwise and anticlockwise replicated arms of the chromosome within a single ring, as shown in a, then completion of chromosome transfer should present no problems. Directionality would need to be determined but movement of the two duplexes in the same direction could be an intrinsic property of the transporter. From a mechanistic point of view, it seems more likely that a ring can accommodate only a single duplex, as shown in b. This would present a topological barrier to the completion of chromosome transfer, which would require resolution, perhaps by ring fusion. However, it is perhaps unrealistic to suppose that the chromosome crosses the septum only twice. How is chromosome transfer completed if, as shown in c and d, the chromosome crosses the septum more than twice? This argues that SpoIIIE rings might be dynamic structures that can assemble and disassemble or fuse, as DNA loops are first encountered in the closing septum and then resolved. Moreover, control of the directionality of transfer becomes potentially even more important. In principle, this could be solved if SpoIIIE is orientated in the membrane such that it pumps in one direction only (c). Alternatively, the direction of transfer could depend on the orientation of the trapped segment (d). The latter mechanism could account better for the role of SpoIIIE at medial division sites where it is anticipated that two chromosomes are moved in opposite directions.

the transport complex that spans the envelopes of the donor and the recipient cells. The structure of TrwB is also exciting because it indicates that, despite relatively low sequence identity, several proteins implicated in DNA metabolism, including the homologous recombination protein RecA, the hexameric helicase T7 gene 4 protein and the δ′ clamp loader subunit of DNA polymerase holoenzyme, might share a common fold41–43. Electron microscopic studies have revealed that some of these proteins can assemble into hexameric ring structures with a central cavity large enough to accommodate DNA (ssDNA in the case of the helicase T7 gene 4 protein and the δ′ clamp loader subunit). It is tempting to include SpoIIIE in the same group of proteins, both because of weak sequence similarity between SpoIIIE and TrwB, and because preliminary results from our laboratory are consistent with the idea that the active carboxy-terminal domain of SpoIIIE forms a hexameric ring through which DNA is transported (J.B., unpublished data). If this disparate group of proteins turns out to be related in structure, it seems likely that they will also operate by similar mechanisms. Questions and possible solutions

The biology of SpoIIIE function raises several interesting issues that relate to its biochemical function. The first question is the nature of the DNA substrate on which SpoIIIE acts. As described above, SpoIIIE probably transfers dsDNA, in contrast to the better characterized conjugative DNA transfer systems, which mobilize ssDNA. However, it is possible that SpoIIIE needs to work on more complex substrates. In principle, when the division

septum closes, it must trap at least two DNA duplexes, representing the clockwise and anticlockwise replicated arms of the chromosome. Does this result in formation of two separate hexameric rings, one on each duplex, or can two duplexes be accommodated in a single ring (FIG. 5)? Furthermore, it is conceivable that the presence of loops in the chromosome will initially result in several duplexes traversing the septum. This topological problem might require the formation of several ring structures and, perhaps, additional mechanisms to merge rings as the loops of DNA they are acting on are reeled in. Further to the problem of how to handle several duplexes, an additional problem — that of the polarity of transfer — is highlighted by this line of reasoning. Given the overall speed of DNA transfer (about 10 to 15 min for about 3 Mbp of DNA14,37,44), and the fact that this apparently occurs against an increasing DNA concentration in the prespore, it seems likely that the DNA transfer machinery must act in a processive and directional manner. In principle, SpoIIIE could be targeted to the septum so that its active site specifically directs DNA transfer in the appropriate direction. However, it is not clear how this asymmetry could be achieved. Furthermore, this kind of model would be difficult to adapt to SpoIIIE-like proteins functioning at medial division sites where two chromosomes might have to be moved in opposite directions. Many bacterial genomes have significantly skewed sequence compositions in the two arms of their chromosomes45. In principle, this sequence asymmetry could provide a mechanism by which to impose directionality on chromosome transfer. Either SpoIIIE could be loaded onto DNA in the appropriate direction by recognition of short asymmetrical sequences, or the sequence asymmetry could bias the direction of an otherwise random mechanism so that transfer in the appropriate direction is favoured. Finally, SpoIIIE seems to have yet another function — determining the proper compartmentalization of transcription between prespore and mother cell during sporulation1,46. Among other issues, this raises the question as to the fate of DNA-binding proteins associated with the chromosome that is transferred into the prespore. Are these proteins stripped off the chromosome as it is pumped into the prespore, or do they remain associated with the DNA and therefore get transferred between the cells along with their binding sites? It is not unrealistic to propose that SpoIIIE is strong enough to displace proteins from DNA because helicases have been shown to displace histones from DNA and even streptavidin from a biotinylated oligonucleotide47. Studies of several members of the SpoIIIE protein superfamily should soon provide a unifying view of their general function and the biochemical basis for DNA transport. A combination of crystallography and advanced biochemical methods, particularly single-molecule methods, should facilitate the attainment of this goal. Links DATABASE LINKS SpoIIIE | ftsK | XerC | CodV | RipX FURTHER INFORMATION ProDom web site | Errington lab


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Acknowledgements Work in the Errington lab is supported by grants from the BBSRC. J.E. acknowledges receipt of a BBSRC Senior Research Fellowship.

