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

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.

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Simple topology: FtsK-directed recombination at the dif site

Biochemical Society Transactions ◽

10.1042/bst20120299 ◽

2013 ◽

Vol 41 (2) ◽

pp. 595-600 ◽

Cited By ~ 11

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.

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The Nucleoid Occlusion Protein SlmA Binds to Lipid Membranes

mBio ◽

10.1128/mbio.02094-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Miguel Ángel Robles-Ramos ◽

William Margolin ◽

Marta Sobrinos-Sanguino ◽

Carlos Alfonso ◽

Germán Rivas ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Dna Binding ◽

Division Ring ◽

Binding Sites ◽

Lipid Membranes ◽

Fine Tuning ◽

Chromosomal Dna ◽

Content Type ◽

Binding Partners

ABSTRACT Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA’s ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly. IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid membranes, adding a novel putative mechanism for regulating the local activity of these cell division proteins. We find that interaction between SlmA and lipid membranes is regulated by SlmA’s DNA binding sites and protein binding partners as well as chemical conditions, suggesting that the SlmA-membrane interactions are important for fine-tuning the regulation of nucleoid integrity during cytokinesis.

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The Escherichia coli DNA translocase FtsK

Biochemical Society Transactions ◽

10.1042/bst0380395 ◽

2010 ◽

Vol 38 (2) ◽

pp. 395-398 ◽

Cited By ~ 47

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.

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Genes Required for Aerial Growth, Cell Division, and Chromosome Segregation Are Targets of WhiA before Sporulation in Streptomyces venezuelae

mBio ◽

10.1128/mbio.00684-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 61

Author(s):

Matthew J. Bush ◽

Maureen J. Bibb ◽

Govind Chandra ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Regulatory Networks ◽

Liquid Culture ◽

Biological Processes ◽

Streptomyces Venezuelae ◽

Content Type ◽

Master Regulators ◽

Aerial Growth ◽

Genes Encoding

ABSTRACTWhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacteriumStreptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA inStreptomycesdevelopment and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus,Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed thatwhiAis required for the initiation of sporulation septation and chromosome segregation inS. venezuelae, and several genes encoding key proteins of theStreptomycescell division machinery, such asftsZ,ftsW, andftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor σWhiGand the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, andfilP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.IMPORTANCESince the initial identification of the genetic loci required forStreptomycesdevelopment, all of thebldandwhidevelopmental master regulators have been cloned and characterized, and significant progress has been made toward understanding the cell biological processes that drive morphogenesis. A major challenge now is to connect the cell biological processes and the developmental master regulators by dissecting the regulatory networks that link the two. Studies of these regulatory networks have been greatly facilitated by the recent introduction ofStreptomyces venezuelaeas a new model system for the genus, a species that sporulates in liquid culture. Taking advantage ofS. venezuelae, we have characterized the regulon of genes directly under the control of one of these master regulators, WhiA. Our results implicate WhiA in the direct regulation of key steps in sporulation, including the cessation of aerial growth, the initiation of cell division, and chromosome segregation.

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Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽

10.1128/mbio.00143-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Yanyu Zhu ◽

Mainak Mustafi ◽

James C. Weisshaar

Keyword(s):

Escherichia Coli ◽

Fluorescence Microscopy ◽

Rna Polymerase ◽

Stationary Phase ◽

Exponential Growth ◽

Mean Square Displacement ◽

Quiescent State ◽

Chromosomal Dna ◽

Content Type ◽

E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

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Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives inEscherichia coliwith Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01041-19 ◽

2019 ◽

Vol 63 (9) ◽

Cited By ~ 1

Author(s):

Sabine Schuster ◽

Martina Vavra ◽

Winfried V. Kern

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Drug Transport ◽

Binding Pocket ◽

Entrance Channel ◽

Transport Pathways ◽

Double Mutation ◽

Content Type ◽

Rnd Transporter ◽

The Impact

ABSTRACTA major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives inEscherichia coliwas confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

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

Journal of Bacteriology ◽

10.1128/jb.182.14.3965-3971.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 3965-3971 ◽

Cited By ~ 98

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.

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The Dimerization Function of MinC Resides in a Structurally Autonomous C-Terminal Domain

Journal of Bacteriology ◽

10.1128/jb.183.22.6684-6687.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6684-6687 ◽

Cited By ~ 27

Author(s):

Tim H. Szeto ◽

Susan L. Rowland ◽

Glenn F. King

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Limited Proteolysis ◽

Terminal Domain ◽

Cell Division Inhibitor

ABSTRACT Limited proteolysis of the Escherichia coli cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain. We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.

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Comparative Genomic Analysis of Third-Generation-Cephalosporin-Resistant Escherichia coli Harboring the blaCMY-2-Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02385-19 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 2

Author(s):

Takahiro Shirakawa ◽

Tsuyoshi Sekizuka ◽

Makoto Kuroda ◽

Satowa Suzuki ◽

Manao Ozawa ◽

...

Keyword(s):

Escherichia Coli ◽

Phylogenetic Analysis ◽

Gene Clustering ◽

Comparative Genomic ◽

Third Generation ◽

Chromosomal Dna ◽

In Ovo ◽

Content Type ◽

E Coli ◽

Network Analyses

ABSTRACT The off-label use of third-generation cephalosporins (3GCs) during in ovo vaccination or vaccination of newly hatched chicks has been a common practice worldwide. CMY-2-producing Escherichia coli strains have been disseminated in broiler chicken production. The objective of this study was to determine the epidemiological linkage of blaCMY-2-positive plasmids among broilers both within and outside Japan, because the grandparent stock and parent stock were imported into Japan. We examined the whole-genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002 to 2014. The predominant 3GC resistance gene was blaCMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (n = 21; 24.1%), IncI (n = 12; 13.8%), IncB/O/K/Z (n = 28; 32.2%), and IncC (n = 22; 25.3%). Those plasmids were subjected to gene clustering, network analyses, and plasmid multilocus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single-nucleotide variant (SNV)-based phylogenetic analysis. MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. The sequence type 429 (ST429) cluster harboring blaCMY-2-positive IncB/O/K/Z was closely related to isolates from broilers in Germany harboring blaCMY-2-positive IncB/O/K/Z. pST55-IncI, pST12-IncI1-Iγ, and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to plasmids detected in E. coli isolates from chickens in North America, whereas 26 IncB/O/K/Z types were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria inside and outside Japan.

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