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.     

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.     

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.     

Cell-specific SpoIIIE assembly and DNA translocation polarity are dictated by chromosome orientation

SpoIIIE and FtsK are related proteins that translocate chromosomes across septa. Previous results suggested that SpoIIIE exports DNA and that translocation polarity is governed by the cell-specific regulation of its assembly, but that FtsK is a reversible motor for which translocation polarity is governed by its DNA substrate. Seeking to reconcile these conclusions, we used cell-specific GFP tagging to demonstrate that SpoIIIE assembles a complex only in the mother cell, from which DNA is exported, but that DNA translocation-defective SpoIIIE proteins assemble in both cells. Altering chromosome architecture by soj-spo0J and racA soj-spo0J mutations allowed wild-type SpoIIIE to assemble in the forespore and export the forespore chromosome. Combining LacI-CFP tagging of oriC with time-lapse microscopy, we demonstrate that the chromosome is exported from the forespore when oriC fails to be trapped in the forespore. Thus, the position of oriC after septation determines which cell will receive the chromosome and which will assemble SpoIIIE.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase

Bacteria harbouring circular chromosomes have a Xer site-specific recombination system that resolves chromosome dimers at division. In Escherichia coli, the activity of the XerCD/dif system is controlled and coupled with cell division by the FtsK DNA translocase. Most Xer systems, as XerCD/dif, include two different recombinases. However, some, as the Lactococcus lactis XerS/dif_SL system, include only one recombinase. We investigated the functional effects of this difference by studying the XerS/dif_SL system. XerS bound and recombined dif_SL sites in vitro, both activities displaying asymmetric characteristics. Resolution of chromosome dimers by XerS/dif_SL required translocation by division septum-borne FtsK. The translocase domain of L. lactis FtsK supported recombination by XerCD/dif, just as E. coli FtsK supports recombination by XerS/dif_SL. Thus, the FtsK-dependent coupling of chromosome segregation with cell division extends to non-rod-shaped bacteria and outside the phylum Proteobacteria. Both the XerCD/dif and XerS/dif_SL recombination systems require the control activities of the FtsKγ subdomain. However, FtsKγ activates recombination through different mechanisms in these two Xer systems. We show that FtsKγ alone activates XerCD/dif recombination. In contrast, both FtsKγ and the translocation motor are required to activate XerS/dif_SL recombination. These findings have implications for the mechanisms by which FtsK activates recombination.     

Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis.

The 787 amino acid SpoIIIE protein of Bacillus subtilis is required for chromosome partitioning during sporulation. This process differs from vegetative chromosome partitioning in that it occurs after formation of the septum, apparently by transfer of the chromosome through the nascent septum in a manner reminiscent of plasmid conjugation. Here we show that SpoIIIE is associated with the cell membrane, with its soluble C-terminal domain located inside the cell. Immunofluorescence microscopy using affinity-purified anti-SpoIIIE antibodies shows that SpoIIIE is targeted near the centre of the asymmetric septum, in support of a direct role for SpoIIIE in transport of DNA through the septum. We also report on the isolation of a mutation affecting the N-terminal hydrophobic domain of SpoIIIE that interferes with targeting to the septum and blocks DNA transfer. This mutation also causes de-localization of the activity of the normally prespore-specific sigma factor, sigmaF, consistent with the notion that SpoIIIE can form a seal between the chromosomal DNA and the leading edge of the division septum.     

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.     

Bacterial translocation motors investigated by single molecule techniques

Translocation of DNA and protein fibers through narrow constrictions is a ubiquitous and crucial activity of bacterial cells. Bacteria use specialized machines to support macromolecular movement. A very important step toward a mechanistic understanding of these translocation machines is the characterization of their physical properties at the single molecule level. Recently, four bacterial transport processes have been characterized by nanomanipulation at the single molecule level, DNA translocation by FtsK and SpoIIIE, DNA import during transformation, and the related process of a type IV pilus retraction. With all four processes, the translocation rates, processivity, and stalling forces were remarkably high as compared with single molecule experiments with other molecular motors. Although substrates of all four processes proceed along a preferential direction of translocation, directionality has been shown to be controlled by distinct mechanisms.     

Separation of chromosome termini during sporulation of Bacillus subtilis depends on SpoIIIE

Bacillus subtilis undergoes a highly distinctive division during spore formation. It yields two unequal cells, the mother cell and the prespore, and septum formation is completed before the origin-distal 70% of the chromosome has entered the smaller prespore. The mother cell subsequently engulfs the prespore. Two different probes were used to study the behavior of the terminus (ter) region of the chromosome during spore formation. Only one ter region was observed at the time of sporulation division. A second ter region, indicative of chromosome separation, was not distinguishable until engulfment was nearing completion, when one was in the mother cell and the other in the prespore. Separation of the two ter regions depended on the DNA translocase SpoIIIE. It is concluded that SpoIIIE is required during spore formation for chromosome separation as well as for translocation; SpoIIIE is not required for separation during vegetative growth.     

Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE

Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix α3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central ～3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.     

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.     

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.