Simple topology: FtsK-directed recombination at the dif site

2013 ◽  
Vol 41 (2) ◽  
pp. 595-600 ◽  
Author(s):  
Ian Grainge
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Motor Domain ◽  
Site Specific ◽  
Multifunctional Protein ◽  
Supercoiled Plasmid ◽  
C Terminus ◽  
Dna Translocase

FtsK is a multifunctional protein, which, in Escherichia coli, co-ordinates the essential functions of cell division, DNA unlinking and chromosome segregation. Its C-terminus is a DNA translocase, the fastest yet characterized, which acts as a septum-localized DNA pump. FtsK's C-terminus also interacts with the XerCD site-specific recombinases which act at the dif site, located in the terminus region. The motor domain of FtsK is an active translocase in vitro, and, when incubated with XerCD and a supercoiled plasmid containing two dif sites, recombination occurs to give unlinked circular products. Despite years of research the mechanism for this novel form of topological filter remains unknown.

The Escherichia coli DNA translocase FtsK

2010 ◽  
Vol 38 (2) ◽  
pp. 395-398 ◽  
Author(s):  
David J. Sherratt ◽  
Lidia K. Arciszewska ◽  
Estelle Crozat ◽  
James E. Graham ◽  
Ian Grainge
Keyword(s):  
Escherichia Coli ◽  
Chromosome Segregation ◽  
Regulatory Domain ◽  
Biological Functions ◽  
Comparable Rate ◽  
Site Specific ◽  
Late Stages ◽  
Dna Translocase

Escherichia coli FtsK is a septum-located DNA translocase that co-ordinates the late stages of cytokinesis and chromosome segregation. Relatives of FtsK are present in most bacteria; in Bacillus subtilis, the FtsK orthologue, SpoIIIE, transfers the majority of a chromosome into the forespore during sporulation. DNA translocase activity is contained within a ~ 512-amino-acid C-terminal domain, which is divided into three subdomains: α, β and γ. α and β comprise the translocation motor, and γ is a regulatory domain that interacts with DNA and with the XerD recombinase. In vitro rates of translocation of ~ 5 kb·s−1 have been measured for both FtsK and SpoIIIE, whereas, in vivo, SpoIIIE has a comparable rate of translocation. Translocation by both of these proteins is not only rapid, but also directed by DNA sequence. This directionality requires interaction of the γ subdomain with specific 8 bp DNA asymmetric sequences that are oriented co-directionally with replication direction of the bacterial chromosome. The γ subdomain also interacts with the XerCD site-specific recombinase to activate chromosome unlinking by recombination at the chromosomal dif site. In the present paper, the properties in vivo and in vitro of FtsK and its relatives are discussed in relation to the biological functions of these remarkable enzymes.

scholarly journals The N-Terminal Membrane-Spanning Domain of the Escherichia coli DNA Translocase FtsK Hexamerizes at Midcell

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Paola Bisicchia ◽  
Bradley Steel ◽  
Mekdes H. Mariam Debela ◽  
Jan Löwe ◽  
David Sherratt
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Chromosomal Dna ◽  
Content Type ◽  
Terminal Domain ◽  
Membrane Spanning ◽  
Late Stages ◽  
Dna Translocase

ABSTRACTBacterial FtsK plays a key role in coordinating cell division with the late stages of chromosome segregation. The N-terminal membrane-spanning domain of FtsK is required for cell division, whereas the C-terminal domain is a fast double-stranded DNA (dsDNA) translocase that brings the replication termination region of the chromosome to midcell, where it facilitates chromosome unlinking by activating XerCD-difsite-specific recombination. Therefore, FtsK coordinates the late stages of chromosome segregation with cell division. Although the translocase is known to act as a hexamer on DNA, it is unknown when and how hexamers form, as is the number of FtsK molecules in the cell and within the divisome. Using single-molecule live-cell imaging, we show that newbornEscherichia colicells growing in minimal medium contain ~40 membrane-bound FtsK molecules that are largely monomeric; the numbers increase proportionately with cell growth. After recruitment to the midcell, FtsK is present only as hexamers. Hexamers are observed in all cells and form before any visible sign of cell constriction. An average of 7 FtsK hexamers per cell are present at midcell, with the N-terminal domain being able to hexamerize independently of the translocase. Detergent-solubilized and purified FtsK N-terminal domains readily form hexamers, as determined byin vitrobiochemistry, thereby supporting thein vivodata. The hexameric state of the FtsK N-terminal domain at the division site may facilitate assembly of a functional C-terminal DNA translocase on chromosomal DNA.IMPORTANCEIn the rod-shaped bacteriumEscherichia coli, more than a dozen proteins act at the cell center to mediate cell division, which initiates while chromosome replication and segregation are under way. The protein FtsK coordinates cell division with the late stages of chromosome segregation. The N-terminal part of FtsK is membrane embedded and acts in division, while the C-terminal part forms a hexameric ring on chromosomal DNA, which the DNA can translocate rapidly to finalize chromosome segregation. Using quantitative live-cell imaging, which measures the position and number of FtsK molecules, we show that in all cells, FtsK hexamers form only at the cell center at the initiation of cell division. Furthermore, the FtsK N-terminal portion forms hexamers independently of the C-terminal translocase.

scholarly journals Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

1998 ◽  
Vol 180 (23) ◽  
pp. 6424-6428 ◽  
Author(s):  
Xuan-Chuan Yu ◽  
Elizabeth K. Weihe ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Null Mutation ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Chromosome Partitioning ◽  
C Terminus

ABSTRACT FtsK is essential for Escherichia coli cell division. We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with amukB null mutation resulted in a synthetic lethal phenotype. When the truncated ftsK was combined with aminCDE deletion, chains of minicells were generated, many of which contained DNA. These results suggest that the C terminus of FtsK has an important role in chromosome partitioning.

scholarly journals Characterization of the Chromosome Dimer Resolution Site in Caulobacter crescentus

2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Ali Farrokhi ◽  
Hua Liu ◽  
George Szatmari
Keyword(s):  
Cell Division ◽  
Caulobacter Crescentus ◽  
Consensus Sequence ◽  
Markov Modeling ◽  
Control Of Gene Expression ◽  
Site Specific ◽  
Content Type ◽  
Site Specific Recombination ◽  
Resolution System

ABSTRACT Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia coli dif site in vitro. Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC, which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology. IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus. Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

scholarly journals Cell Division Protein FtsZ Is Unfolded for N-Terminal Degradation by Antibiotic-Activated ClpP

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Nadine Silber ◽  
Stefan Pan ◽  
Sina Schäkermann ◽  
Christian Mayer ◽  
Heike Brötz-Oesterhelt ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Unfolding ◽  
Multidrug Resistant ◽  
Unexpected Finding ◽  
Bacterial Cells ◽  
Clp Protease ◽  
Content Type ◽  
C Terminus ◽  
Cell Division Protein

ABSTRACT Antibiotic acyldepsipeptides (ADEPs) deregulate ClpP, the proteolytic core of the bacterial Clp protease, thereby inhibiting its native functions and concomitantly activating it for uncontrolled proteolysis of nonnative substrates. Importantly, although ADEP-activated ClpP is assumed to target multiple polypeptide and protein substrates in the bacterial cell, not all proteins seem equally susceptible. In Bacillus subtilis, the cell division protein FtsZ emerged to be particularly sensitive to degradation by ADEP-activated ClpP at low inhibitory ADEP concentrations. In fact, FtsZ is the only bacterial protein that has been confirmed to be degraded in vitro as well as within bacterial cells so far. However, the molecular reason for this preferred degradation remained elusive. Here, we report the unexpected finding that ADEP-activated ClpP alone, in the absence of any Clp-ATPase, leads to an unfolding and subsequent degradation of the N-terminal domain of FtsZ, which can be prevented by the stabilization of the FtsZ fold via nucleotide binding. At elevated antibiotic concentrations, importantly, the C terminus of FtsZ is notably targeted for degradation in addition to the N terminus. Our results show that different target structures are more or less accessible to ClpP, depending on the ADEP level present. Moreover, our data assign a Clp-ATPase-independent protein unfolding capability to the ClpP core of the bacterial Clp protease and suggest that the protein fold of FtsZ may be more flexible than previously anticipated. IMPORTANCE Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 236-243 ◽  
Author(s):  
Daisuke Shiomi ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Additional Function ◽  
Ring Assembly ◽  
C Terminus ◽  
Min Proteins ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

1998 ◽  
Vol 180 (23) ◽  
pp. 6424-6428 ◽  
Author(s):  
Xuan-Chuan Yu ◽  
Elizabeth K. Weihe ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Chromosome Segregation ◽  
C Terminus

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli

PLoS Genetics ◽  
2017 ◽  
Vol 13 (3) ◽  
pp. e1006702 ◽  
Author(s):  
Elisa Galli ◽  
Caroline Midonet ◽  
Evelyne Paly ◽  
François-Xavier Barre
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Growth Conditions ◽  
Fast Growth
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