The ATPase SpoIIIE transports DNA across fused septal membranes during sporulation in Bacillus subtilis

The FtsK/SpoIIIE family of ATP-dependent DNA transporters mediates proper chromosome segregation in dividing bacteria. In sporulating Bacillus subtilis cells, SpoIIIE translocates much of the circular chromosome from the mother cell into the forespore, but the molecular mechanism remains unclear. Using a new assay to monitor DNA transport, we demonstrate that the two arms of the chromosome are simultaneously pumped into the forespore. Up to 70 molecules of SpoIIIE are recruited to the site of DNA translocation and assemble into complexes that could contain 12 subunits. The fusion of the septal membranes during cytokinesis precedes DNA translocation and does not require SpoIIIE, as suggested by analysis of lipid dynamics, serial thin-section electron microscopy, and cell separation by protoplasting. These data support a model for DNA transport in which the transmembrane segments of FtsK/SpoIIIE form linked DNA-conducting channels across the two lipid bilayers of the septum.     

FtsK and SpoIIIE: the tale of the conserved tails

During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The preferential assembly of SpoIIIE complexes in the mother cell provided the idea that SpoIIIE functioned as a DNA exporter, which ensured translocation orientation. In this issue of Molecular Microbiology, Becker and Pogliano reinvestigate the molecular mechanisms that orient the activity of SpoIIIE. Their findings indicate that SpoIIIE reads the polarity of DNA like its Escherichia coli homologue, FtsK.     

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

During Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.     

MinCD-dependent regulation of the polarity of SpoIIIE assembly and DNA transfer

During Bacillus subtilis sporulation, the SpoIIIE DNA translocase moves a trapped chromosome across the sporulation septum into the forespore. The direction of DNA translocation is controlled by the specific assembly of SpoIIIE in the mother cell and subsequent export of DNA into the forespore. We present evidence that the MinCD heterodimer, which spatially regulates cell division during vegetative growth, serves as a forespore-specific inhibitor of SpoIIIE assembly. The deletion of minCD increases the ability of forespore-expressed SpoIIIE to assemble and translocate DNA, and causes otherwise wild-type cells to reverse the direction of DNA transfer, producing anucleate forespores. We propose that two distinct mechanisms ensure the specific assembly of SpoIIIE in the mother cell, the partitioning of more SpoIIIE molecules into the larger mother cell by asymmetric cell division and the MinCD-dependent repression of SpoIIIE assembly in the forespore. Our results suggest that the ability of MinCD to sense positional information is utilized during sporulation to regulate protein assembly differentially on the two faces of the sporulation septum.     

Staphylococcus aureus requires at least one FtsK/SpoIIIE protein for correct chromosome segregation

Faithful coordination between bacterial cell division and chromosome segregation in rod‐shaped bacteria, such as Escherichia coli and Bacillus subtilis, is dependent on the DNA translocase activity of FtsK/SpoIIIE proteins, which move DNA away from the division site before cytokinesis is completed. However, the role of these proteins in chromosome partitioning has not been well studied in spherical bacteria. Here, it was shown that the two Staphylococcus aureus FtsK/SpoIIIE homologues, SpoIIIE and FtsK, operate in independent pathways to ensure correct chromosome management during cell division. SpoIIIE forms foci at the centre of the closing septum in at least 50% of the cells that are close to complete septum synthesis. FtsK is a multifunctional septal protein with a C‐terminal DNA translocase domain that is not required for correct chromosome management in the presence of SpoIIIE. However, lack of both SpoIIIE and FtsK causes severe nucleoid segregation and morphological defects, showing that the two proteins have partially redundant roles in S. aureus.     

SpoIIIE mechanism of directional translocation involves target search coupled to sequence‐dependent motor stimulation

SpoIIIE/FtsK are membrane‐anchored, ATP‐fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence‐recognition modules (SpoIIIE‐γ/FtsK‐γ) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single‐molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single‐molecule and modelling methods that hexameric SpoIIIE binds DNA non‐specifically and finds SRS by an ATP‐independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE‐γ to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS‐directed translocation by SpoIIIE/FtsK.     

Mechanistic Study of Classical Translocation-Dead SpoIIIE36 Reveals the Functional Importance of the Hinge within the SpoIIIE Motor

SpoIIIE/FtsK ATPases are central players in bacterial chromosome segregation. It remains unclear how these DNA translocases harness chemical energy (ATP turnover) to perform mechanical work (DNA movement). Bacillus subtilis sporulation provides a dramatic example of intercompartmental DNA transport, in which SpoIIIE moves 70% of the chromosome across the division plane. To understand the mechanistic requirements for DNA translocation, we investigated the DNA translocation defect of a classical nontranslocating allele, spoIIIE36. We found that the translocation phenotype is caused by a single substitution, a change of valine to methionine at position 429 (V429M), within the motor of SpoIIIE. This substitution is located at the base of a hinge between the RecA-like β domain and the α domain, which is a domain unique to the SpoIIIE/FtsK family and currently has no known function. V429M interferes with both protein-DNA interactions and oligomer assembly. These mechanistic defects disrupt coordination between ATP turnover and DNA interaction, effectively uncoupling ATP hydrolysis from DNA movement. Our data provide the first functional evidence for the importance of the hinge in DNA translocation.     

Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.     

Bacterial chromosome segregation

In most bacteria two vital processes of the cell cycle: DNA replication and chromosome segregation overlap temporally. The action of replication machinery in a fixed location in the cell leads to the duplication of oriC regions, their rapid separation to the opposite halves of the cell and the duplicated chromosomes gradually moving to the same locations prior to cell division. Numerous proteins are implicated in co-replicational DNA segregation and they will be characterized in this review. The proteins SeqA, SMC/MukB, MinCDE, MreB/Mbl, RacA, FtsK/SpoIIIE playing different roles in bacterial cells are also involved in chromosome segregation. The chromosomally encoded ParAB homologs of active partitioning proteins of low-copy number plasmids are also players, not always indispensable, in the segregation of bacterial chromosomes.     

The Bacillus subtilis SftA (YtpS) and SpoIIIE DNA translocases play distinct roles in growing cells to ensure faithful chromosome partitioning

In several bacterial species, the faithful completion of chromosome partitioning is known to be promoted by a conserved family of DNA translocases that includes Escherichia coli FtsK and Bacillus subtilis SpoIIIE. FtsK localizes at nascent division sites during every cell cycle and stimulates chromosome decatenation and the resolution of chromosome dimers formed by recA -dependent homologous recombination. In contrast, SpoIIIE localizes at sites where cells have divided and trapped chromosomal DNA in the membrane, which happens during spore development and under some conditions when DNA replication is perturbed. SpoIIIE completes chromosome segregation post-septationally by translocating trapped DNA across the membrane. Unlike E. coli , B. subtilis contains a second uncharacterized FtsK/SpoIIIE-like protein, SftA (formerly YtpS). We report that SftA plays a role similar to FtsK during each cell cycle but cannot substitute for SpoIIIE in rescuing trapped chromosomes. SftA colocalizes with FtsZ at nascent division sites but not with SpoIIIE at sites of chromosome trapping. SftA mutants divide over unsegregated chromosomes more frequently than wild-type unless recA is inactivated, suggesting that SftA, like FtsK, stimulates chromosome dimer resolution. Having two FtsK/SpoIIIE paralogues is not conserved among endospore-forming bacteria, but is highly conserved within several groups of soil- and plant-associated bacteria.     

SpoIIIE Protein Achieves Directional DNA Translocation through Allosteric Regulation of ATPase Activity by an Accessory Domain

Bacterial chromosome segregation utilizes highly conserved directional translocases of the SpoIIIE/FtsK family. These proteins employ an accessory DNA-binding domain (γ) to dictate directionality of DNA transport. It remains unclear how the interaction of γ with specific recognition sequences coordinates directional DNA translocation. We demonstrate that the γ domain of SpoIIIE inhibits ATPase activity of the motor domain in the absence of DNA but stimulates ATPase activity through sequence-specific DNA recognition. Furthermore, we observe that communication between γ subunits is necessary for both regulatory roles. Consistent with these findings, the γ domain is necessary for robust DNA transport along the length of the chromosome in vivo. Together, our data reveal that directional activation involves allosteric regulation of ATP turnover through coordinated action of γ domains. Thus, we propose a coordinated stimulation model in which γ-γ communication is required to translate DNA sequence information from each γ to its respective motor domain.     

Membrane-associated DNA Transport Machines

DNA pumps play important roles in bacteria during cell division and during the transfer of genetic material by conjugation and transformation. The FtsK/SpoIIIE proteins carry out the translocation of double-stranded DNA to ensure complete chromosome segregation during cell division. In contrast, the complex molecular machines that mediate conjugation and genetic transformation drive the transport of single stranded DNA. The transformation machine also processes this internalized DNA and mediates its recombination with the resident chromosome during and after uptake, whereas the conjugation apparatus processes DNA before transfer. This article reviews these three types of DNA pumps, with attention to what is understood of their molecular mechanisms, their energetics and their cellular localizations.The transport of DNA across membranes by bacteria occurs during sporulation, during cytokinesis, directly from other cells and from the environment. This review addresses the question “how is the DNA polyanion transferred processively across the hydrophobic membrane barrier”?DNA transport must occur through water-filled channels, at least conceptually addressing the problem posed by the hydrophobic membrane. DNA transporters presumably use metabolic energy directly or a coupled-flow (symporter or antiporter) mechanism to drive DNA processively through the channel. It is possible that a Brownian ratchet mechanism, in which directionality is imposed on a diffusive process, also contributes to transport.In this article, we will consider several DNA transport systems. We will begin with the simplest one, namely the FtsK/SpoIIIE system that is involved in cell division and sporulation. We will then turn to the more complex, multiprotein DNA uptake systems that accomplish genetic transformation (the uptake of environmental DNA from the environment) and the conjugation systems of Gram-negative bacteria that mediate the unidirectional transfer of DNA between cells. In each case we will discuss the proteins involved, their actions and the sources of energy that drive transport. Space limitations prevent discussion of other relevant topics, such as DNA transport during bacteriophage infection and more than a brief reference to conjugation in Gram-positive bacteria.     

A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis

The cis-acting sequences required for chromosome segregation are poorly understood in most organisms, including bacteria. Sporulating cells of Bacillus subtilis undergo an unusual asymmetric cell division during which the origin of DNA replication (oriC) region of the chromosome migrates to an extreme polar position. We have now characterized the sequences required for this migration. We show that the previously characterized soj-spo0J chromosome segregation system is not essential for chromosome movement to the cell pole, so this must be driven by an additional segregation mechanism. Observations on a large set of precisely engineered chromosomal inversions and translocations have identified a polar localization region (PLR), which lies approximately 150-300 kbp to the left of oriC. Surprisingly, oriC itself has no involvement in this chromosome segregation system. Dissection of the PLR showed that it has internal functional redundancy, reminiscent of the large diffuse centromeres of most eukaryotic cells.     

Do the same traffic rules apply? Directional chromosome segregation by SpoIIIE and FtsK

Over a decade of studies have tackled the question of how FtsK/SpoIIIE translocases establish and maintain directional DNA translocation during chromosome segregation in bacteria. FtsK/SpoIIIE translocases move DNA in a highly processive, directional manner, where directionality is facilitated by sequences on the substrate DNA molecules that are being transported. In recent years, structural, biochemical, single‐molecule and high‐resolution microscopic studies have provided new insight into the mechanistic details of directional DNA segregation. Out of this body of work, a series of models have emerged and, ultimately, yielded two seemingly opposing models: the loading model and the target search model. We review these recent mechanistic insights into directional DNA movement and discuss the data that may serve to unite these suggested models, as well as propose future directions that may ultimately solve the debate.     

Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis

Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane. Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium. The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell. The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore. As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell. Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore. Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint. We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase. We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission.     

FtsK translocation on DNA stops at XerCD-dif

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKγ regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.     

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.