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.     

Extent of the activity domain and possible roles of FtsK in the Escherichia coli chromosome terminus

Escherichia coli FtsK protein couples cell division and chromosome segregation. It is a component of the septum essential for cell division. It also acts during chromosome dimer resolution by XerCD-specific recombination at the dif site, with two distinct activities: DNA translocation oriented by skewed sequence elements and direct activation of Xer recombination. Dimer resolution requires that the skewed elements polarize in opposite directions 30-50 kb on either side of dif. This constitutes the DIF domain, approximately coincident with the region where replication terminates. The observation that the ftsK1 mutation increases recombination near dif was exploited to determine whether the chromosome region on which FtsK acts is limited to the DIF domain. A monitoring of recombination activity at multiple loci in a 350 kb region to the left of dif revealed (i) zones of differing activities unconnected to dimer resolution and (ii) a constant 10-fold increase of recombination in the 250 kb region adjacent to dif in the ftsK1 mutant. The latter effect allows definition of an FTSK domain whose total size is at least fourfold that of the DIF domain. Additional analyses revealed that FtsK activity responds to polarization in the whole FTSK domain and that displacement of the region where replication terminates preserves differences between recombination zones. Our interpretation is that translocation by FtsK occurs mostly on DNA belonging to a specifically organized domain of the chromosome, when physical links between either dimeric or still intercatenated chromosomes force this DNA to run across the septum at division.     

FtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein

Escherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N-terminal transmembrane domain (FtsK(N)) is essential for septum formation, whereas its C-terminal domain (FtsK(C)) is required for chromosome dimer resolution by XerCD-dif site-specific recombination. FtsK(C) is an ATP-dependent DNA translocase. In vitro and in vivo data point to a dual role for this domain in chromosome dimer resolution (i) to directly activate recombination by XerCD-dif and (ii) to bring recombination sites together and/or to clear DNA from the closing septum. FtsK(N) and FtsK(C) are separated by a long linker region (FtsK(L)) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsK(L) and/or of FtsK(C), of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsK(C). Phenotypic characterization of the mutants indicated a role for FtsK(L) in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsK(C), mutants were found that can perform only one or the other of these two functions, which allowed their separation in vivo for the first time.     

Molecular mechanism of sequence-directed DNA loading and translocation by FtsK

Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.     

Double-stranded DNA translocation: structure and mechanism of hexameric FtsK

FtsK is a DNA translocase that coordinates chromosome segregation and cell division in bacteria. In addition to its role as activator of XerCD site-specific recombination, FtsK can translocate double-stranded DNA (dsDNA) rapidly and directionally and reverse direction. We present crystal structures of the FtsK motor domain monomer, showing that it has a RecA-like core, the FtsK hexamer, and also showing that it is a ring with a large central annulus and a dodecamer consisting of two hexamers, head to head. Electron microscopy (EM) demonstrates the DNA-dependent existence of hexamers in solution and shows that duplex DNA passes through the middle of each ring. Comparison of FtsK monomer structures from two different crystal forms highlights a conformational change that we propose is the structural basis for a rotary inchworm mechanism of DNA translocation.     

FtsK – a bacterial cell division checkpoint?

FtsK is a multifunctional, multidomain protein that acts to co-ordinate chromosome unlinking, segregation and cell division. In this issue of Molecular Microbiology, the report by Dubarry et al. reveals new insight into the surprisingly complex relationship between the different activities of FtsK. The new study makes extensive use of fusion proteins to highlight the role of the FtsK 'linker' domain in helping to co-ordinate these processes. This, taken together with previous studies, suggests that FtsK is intimately involved in septum constriction, physically contacting several other divisome proteins. Further, it is attractive to speculate that FtsK can regulate the late stages of septation to act as a checkpoint to ensure DNA is fully cleared from the septum before it is allowed to close, as well as being the driving force to unlink the chromosomes and segregate the DNA away from the septum.     

The Escherichia coli FtsK functional domains involved in its interaction with its divisome protein partners

FtsK is a multifunctional protein involved in both cell division and chromosome segregation. As far as its role in cell division is concerned, FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components. The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by the two-hybrid assay was already shown by our group. In this work, we describe the identification of the protein domain(s) involved in the interaction with the cell division partner proteins. The biological role of some interactions is also discussed.     

Multiple regions along the Escherichia coli FtsK protein are implicated in cell division

Escherichia coli FtsK is a large 1329 aa integral membrane protein, which links cell division and chromosome segregation through the respective activities of its 200 aa amino-terminal domain, FtsK(N), and its 500 aa carboxy-terminal domain, FtsK(C). A long 600 aa linker, FtsK(L), connects these two domains. Only FtsK(N) is essential for cell division. However, previous observations suggested that the cytoplasmic part of FtsK also participates in the process of septation. Here, we identify two distinct regions within FtsK(L), FtsK(179-331) and FtsK(332-641), which together with FtsK(N), are required for normal septation. We discuss how the implication of multiple regions along the FtsK protein in cell division could participate in the co-ordination of this process with the last stages of chromosome segregation.     

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.     

Membrane topology of the N-terminus of the Escherichia coli FtsK division protein

The Escherichia coli FtsK protein targets the septum, is essential for cell division and may play a role in DNA partitioning. Computer modelling suggests that the first 180 amino acids of the protein are embedded in the cytoplasmic membrane by up to six transmembrane domains. We demonstrate, using gene fusions, that the N-terminus contains four transmembrane helices that link two periplasmic domains. The first periplasmic domain contains an HEXXH amino acid sequence characteristic of zinc metalloproteases. We show by mutation analysis that the conserved glutamic acid of the HEXXH sequence is essential for FtsK function during septation.     

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK

We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(−)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein–protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (−)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.     

Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK

In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane-associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N-terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.     

Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsK_N) is essential for division, and the C terminus (FtsK_C) is a well characterized DNA translocase. Although the function of FtsK_N is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsK_N and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsK_N is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsK_N was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsK_N also provides an important platform for future studies on essential interactions required for this process.     

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double‐stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.     

Asymmetric activation of Xer site-specific recombination by FtsK

Chromosome dimers, which frequently form in Escherichia coli, are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific site on the chromosome, dif, together with the cell division protein FtsK. The C-terminal domain of FtsK (FtsK(C)) is a DNA translocase implicated in helping synapsis of the dif sites and in locally promoting XerD strand exchanges after synapse formation. Here we show that FtsK(C) ATPase activity is directly involved in the local activation of Xer recombination at dif, by using an intermolecular recombination assay that prevents significant DNA translocation, and we confirm that FtsK acts before Holliday junction formation. We show that activation only occurs with a DNA segment adjacent to the XerD-binding site of dif. Only one such DNA extension is required. Taken together, our data suggest that FtsK needs to contact the XerD recombinase to switch its activity on using ATP hydrolysis.     

Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment

Escherichia coli FtsK is an essential cell division protein, which is thought to pump chromosomal DNA through the closing septum in an oriented manner by following DNA sequence polarity. Here, we perform single-molecule measurements of translocation by FtsK50C, a derivative that functions as a DNA translocase in vitro. FtsK50C translocation follows Michaelis-Menten kinetics, with a maximum speed of approximately 6.7 kbp/s. We present results on the effect of applied force on the speed, distance translocated, and the mean times during and between protein activity. Surprisingly, we observe that FtsK50C can spontaneously reverse its translocation direction on a fragment of E. coli chromosomal DNA, indicating that DNA sequence is not the sole determinant of translocation direction. We conclude that in vivo polarization of FtsK translocation could require the presence of cofactors; alternatively, we propose a model in which tension in the DNA directs FtsK translocation.     

FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.     

Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging

Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK–HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK–HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal–bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the ‘genomic context’ combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK–HerA superfamily ATPases.     

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.     

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.