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

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.

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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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Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

mBio ◽

10.1128/mbio.01580-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 1

Author(s):

Markus Peschke ◽

Mélanie Le Goff ◽

Gregory M. Koningstein ◽

Norbert O. Vischer ◽

Abbi Abdel-Rehim ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Transmembrane Domain ◽

Signal Recognition Particle ◽

Membrane Insertion ◽

Type Ii ◽

Content Type ◽

E Coli ◽

C Terminus

ABSTRACT Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented. IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus “regular” membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

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Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

Journal of Cell Science ◽

10.1242/jcs.99.4.823 ◽

1991 ◽

Vol 99 (4) ◽

pp. 823-836

Author(s):

S.J. Atkinson ◽

M. Stewart

Keyword(s):

Escherichia Coli ◽

Ionic Strength ◽

Coiled Coil ◽

Periodic Variation ◽

Heptad Repeat ◽

C Terminus ◽

N Terminus ◽

Low Ionic Strength ◽

Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

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Quantitative Investigation of the Role of SeqA in Escherichia Coli Chromosome Segregation

Biophysical Journal ◽

10.1016/j.bpj.2014.11.2168 ◽

2015 ◽

Vol 108 (2) ◽

pp. 395a

Author(s):

Julie A. Cass ◽

Nathan J. Kuwada ◽

Paul A. Wiggins

Keyword(s):

Escherichia Coli ◽

Chromosome Segregation ◽

Quantitative Investigation

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Enzymic and Chemical Reduction of the Iron Center of the Escherichia coli Ribonucleotide Reductase Protein R2. The Role of the C-Terminus

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.357_1.x ◽

1995 ◽

Vol 233 (1) ◽

pp. 357-363 ◽

Cited By ~ 15

Author(s):

Jacques Coves ◽

Betty Delon ◽

Isabel Climent ◽

Britt-Marie Sjoberg ◽

Marc Fontecave

Keyword(s):

Escherichia Coli ◽

Ribonucleotide Reductase ◽

Chemical Reduction ◽

C Terminus ◽

Iron Center

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Chemotaxis of Escherichia coli to Pyrimidines: a New Role for the Signal Transducer Tap

Journal of Bacteriology ◽

10.1128/jb.01590-07 ◽

2007 ◽

Vol 190 (3) ◽

pp. 972-979 ◽

Cited By ~ 36

Author(s):

Xianxian Liu ◽

Rebecca E. Parales

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Growth Conditions ◽

Wild Type ◽

E Coli ◽

C Terminus ◽

Signaling Domain ◽

Chemotaxis Proteins ◽

Periplasmic Domain

ABSTRACT Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

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Coregulated assembly of actin-like FtsA polymers with FtsZ during Z-ring formation and division in Escherichia coli

10.1101/2021.01.12.426377 ◽

2021 ◽

Author(s):

Josiah J. Morrison ◽

Joseph Conti ◽

Jodi L. Camberg

Keyword(s):

Escherichia Coli ◽

Atp Hydrolysis ◽

Direct Interaction ◽

Ring Formation ◽

Phospholipid Vesicle ◽

E Coli ◽

C Terminus ◽

Z Ring

AbstractIn Escherichia coli, the actin homolog FtsA localizes the cell division machinery, beginning with the Z-ring, to the cytoplasmic membrane through direct interaction with FtsZ. FtsZ polymers are first to assemble at the Z-ring at midcell, where they direct constriction and septation. While FtsZ polymerization is critical for establishing a functional Z-ring that leads to constriction, the assembly state of FtsA and the role of FtsA ATP utilization during division in E. coli remain unclear. Here, we show that ATP hydrolysis, FtsZ interaction, and phospholipid vesicle remodeling by FtsA are impaired by a substitution mutation at the predicted active site for hydrolysis. This mutation, Glu 14 to Arg, also impairs Z-ring assembly and division in vivo. To further investigate the role of phospholipid engagement and ATP utilization in regulating FtsA function, we characterized a truncated E. coli FtsA variant, FtsA(ΔMTS), which lacks the region at the C-terminus important for engaging the membrane and is defective for ATP hydrolysis. We show that E. coli FtsA(ΔMTS) forms ATP-dependent actin-like filaments and assembly is antagonized by FtsZ. Polymerization of FtsZ with GTP, or a non-hydrolyzable analog, blocks inhibition of ATP-dependent FtsA assembly, and instead favors coassembly of stable FtsA/FtsZ polymers. In the cell, FtsA/FtsZ coassembly is favored at midcell, where FtsZ polymerizes, and inhibited at regions where FtsZ polymers are destabilized by regulators, such as MinC at the poles or SlmA at the nucleoid. We show that MinC prevents recruitment of FtsZ, via FtsA, to phospholipids, suggesting that local interactions of MinC with FtsZ block membrane tethering and uncouple the Z-ring from its major membrane contact. During Z-ring formation, the coassembly of FtsZ polymers with FtsA is coordinated and is a critical early step in division. This step also serves as a checkpoint by responding to the suite of FtsZ assembly regulators in the cell that modulate Z-ring position and dynamics prior to initiating cell wall synthesis.

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Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

Essays in Biochemistry ◽

10.1042/ebc20190080 ◽

2020 ◽

Vol 64 (2) ◽

pp. 251-261

Author(s):

Jessica E. Fellmeth ◽

Kim S. McKim

Keyword(s):

Chromosome Segregation ◽

New Technologies ◽

Early Prophase ◽

Unique Role ◽

Kinetochore Assembly ◽

M Phase ◽

In Vivo Analysis ◽

Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650369 ◽

1996 ◽

Vol 75 (05) ◽

pp. 796-800 ◽

Cited By ~ 10

Author(s):

Sanne Valentin ◽

Inger Schousboe

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Microtitre Plate ◽

C Terminus ◽

Microtitre Plate Assay ◽

Kunitz Domain ◽

Tissue Factor Pathway ◽

Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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