scholarly journals The Escherichia coli datA site promotes proper regulation of cell division

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 703-710 ◽  
Author(s):  
Morigen Morigen ◽  
Ingvild Flåtten ◽  
Kirsten Skarstad
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Binding Sites ◽  
Replication Initiation ◽  
Permissive Temperature ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Initiator

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA. An excess of datA sites provided on plasmids has been found to lead to both a delay in initiation of replication and in cell division during exponential growth. Here we have investigated the effect of datA on the cell division block that occurs upon inhibition of replication initiation in a dnaC2 mutant. We found that this checkpoint mechanism was aided by the presence of datA. In cells where datA was deleted or an excess of DnaA was provided, cell division occurred in the absence of replication and anucleate cells were formed. This finding indicates that loss of datA and/or excess of DnaA protein promote cell division. This conclusion was supported by the finding that the lethality of the division-compromised mutants ftsZ84 and ftsI23 was suppressed by deletion of datA, at the lowest non-permissive temperature. We propose that the cell division block that occurs upon inhibition of DNA replication is, at least in part, due to a drop in the concentration of the ATP–DnaA protein.

scholarly journals Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

2007 ◽  
Vol 189 (23) ◽  
pp. 8660-8666 ◽  
Author(s):  
Henrik J. Nielsen ◽  
Brenda Youngren ◽  
Flemming G. Hansen ◽  
Stuart Austin
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Replication Forks ◽  
Daughter Cells ◽  
Initiation Of Replication ◽  
Replication Terminus ◽  
Multiple Replication

ABSTRACT Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division, the chromosomes contain multiple replication forks and must be segregated while this complex pattern of replication is still ongoing. Here, we show that replication and segregation continue in step, starting at the origin and progressing to the replication terminus. Thus, early-replicated markers on the multiple-branched chromosomes continue to separate soon after replication to form separate protonucleoids, even though they are not segregated into different daughter cells until later generations. The segregation pattern follows the pattern of chromosome replication and does not follow the cell division cycle. No extensive cohesion of sister DNA regions was seen at any growth rate. We conclude that segregation is driven by the progression of the replication forks.

scholarly journals Degradation of mutant initiator protein DnaA204 by proteases ClpP, ClpQ and Lon is prevented when DNA is SeqA-free

2003 ◽  
Vol 370 (3) ◽  
pp. 867-871 ◽  
Author(s):  
Monika SLOMINSKA ◽  
Anne WAHL ◽  
Grzegorz WEGRZYN ◽  
Kirsten SKARSTAD
Keyword(s):  
Escherichia Coli ◽  
Lon Protease ◽  
Mutant Form ◽  
Dna Complexes ◽  
Initiator Protein ◽  
Dnaa Protein ◽  
Replication Initiator Protein ◽  
Initiation Frequency ◽  
Replication Initiator ◽  
Mutant Cells

A mutant form of the Escherichia coli replication initiator protein, DnaA204, is unstable. At low growth rates, the dnaA204 mutant cells experience a limitation of initiator protein and grow with reduced initiation frequency and DNA concentration. The mutant DnaA protein is stabilized by the lack of SeqA protein. This stabilization was also observed in a dam mutant where the chromosome remains unmethylated. Since unmethylated DNA is not bound by SeqA, this indicates that DnaA204 is not stabilized by the lack of SeqA protein by itself, but rather by lack of SeqA complexed with DNA. Thus the destabilization of DnaA204 may be due either to interaction with SeqA—DNA complexes or changes in nucleoid organization and superhelicity caused by SeqA. The DnaA204 protein was processed through several chaperone/protease pathways. The protein was stabilized by the presence of the chaperones ClpA and ClpX and degraded by their cognate protease ClpP. The dnaA204 mutant was not viable in the absence of ClpY, indicating that this chaperone is essential for DnaA204 stability or function. Its cognate protease ClpQ, as well as Lon protease, degraded DnaA204 to the same degree as ClpP. The chaperones GroES, GroEL and DnaK contributed to stabilization of DnaA204 protein.

scholarly journals Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

2004 ◽  
Vol 186 (12) ◽  
pp. 3785-3793 ◽  
Author(s):  
T. Betteridge ◽  
J. Yang ◽  
A. J. Pittard ◽  
J. Praszkier
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Origin Of Replication ◽  
Initiator Protein ◽  
Origin Of Dna Replication ◽  
Replication Initiator Protein ◽  
Replication Initiator

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5′-TCTTAA-3′, which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

scholarly journals Dynamic Association of the Replication Initiator and Transcription Factor DnaA with the Bacillus subtilis Chromosome during Replication Stress

2008 ◽  
Vol 191 (2) ◽  
pp. 486-493 ◽  
Author(s):  
Adam M. Breier ◽  
Alan D. Grossman
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Initiation ◽  
Pass Through ◽  
Bacillus Subtilis Chromosome ◽  
Rapid Signaling ◽  
Replication Initiator

ABSTRACT DnaA functions as both a transcription factor and the replication initiator in bacteria. We characterized the DNA binding dynamics of DnaA on a genomic level. Based on cross-linking and chromatin immunoprecipitation data, DnaA binds at least 17 loci, 15 of which are regulated transcriptionally in response to inhibition of replication (replication stress). Six loci, each of which has a cluster of at least nine potential DnaA binding sites, had significant increases in binding by DnaA when replication was inhibited, indicating that the association of DnaA with at least some of its target sites is altered after replication stress. When replication resumed from oriC after inhibition of replication initiation, these high levels of binding decreased rapidly at origin-proximal and origin-distal regions, well before a replication fork could pass through each of the regulated regions. These findings indicate that there is rapid signaling to decrease activation of DnaA during replication and that interaction between DnaA bound at each site and the replication machinery is not required for regulation of DnaA activity in response to replication stress.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter

2009 ◽  
Vol 191 (18) ◽  
pp. 5706-5716 ◽  
Author(s):  
Justine Collier ◽  
Lucy Shapiro
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Regulatory Mechanism ◽  
Caulobacter Crescentus ◽  
Replication Initiation ◽  
Dnaa Protein ◽  
Additional Layer ◽  
Initiation Of Dna Replication ◽  
The Right ◽  
Feedback Regulatory Mechanism

ABSTRACT Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

scholarly journals Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Godefroid Charbon ◽  
Belén Mendoza-Chamizo ◽  
Christopher Campion ◽  
Xiaobo Li ◽  
Peter Ruhdal Jensen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Growth Conditions ◽  
Chromosome Replication ◽  
Replication Initiation ◽  
Atp Depletion ◽  
Initiator Protein ◽  
Cycle Arrest ◽  
A Cell

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

scholarly journals The Nucleoid Occlusion Protein SlmA Binds to Lipid Membranes

mBio ◽  
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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