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Biomedical subjects

Anders Løbner-Olesen

Publications and source records attributed to Anders Løbner-Olesen.

13 recordsLinked to original sources
PubMed2006

Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome.

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.

Adenosine Triphosphatases↗
PubMed2006

Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli.

The actin-like MreB cytoskeletal protein and RNA polymerase (RNAP) have both been suggested to provide the force for chromosome segregation. Here, we identify MreB and RNAP as in vivo interaction partners. The interaction was confirmed using in vitro purified components. We also present convincing evidence that MreB and RNAP are both required for chromosome segregation in Escherichia coli. MreB is required for origin and bulk DNA segregation, whereas RNAP is required for bulk DNA, terminus, and possibly also for origin segregation. Furthermore, flow cytometric analyses show that MreB depletion and inactivation of RNAP confer virtually identical and highly unusual chromosome segregation defects. Thus, our results raise the possibility that the MreB-RNAP interaction is functionally important for chromosome segregation.

Actins↗
PubMed2006

Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB.

Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.

Bacterial Proteins↗
PubMed2005

Dam methylation: coordinating cellular processes.

GATC sequences in Escherichia coli DNA are methylated at the adenine residue by DNA adenine methyltransferase (DamMT). These methylated residues and/or the level of DamMT can influence cellular functions such as gene transcription, DNA mismatch repair, initiation of chromosome replication and nucleoid structure. In certain bacteria, unlike E. coli, DamMT is essential for viability perhaps owing to its role in chromosome replication. DamMT has also been implicated as a virulence factor in bacterial pathogenesis. The origin and phylogeny of DamMT, based on sequenced genomes, has been deduced.

Bacterial Physiological Phenomena↗
PubMed2005

Prokaryotic toxin-antitoxin stress response loci.

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.

Antitoxins↗
PubMed2005

Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant.

The dnaX gene of Escherichia coli encodes tau and gamma clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and tau or gamma is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.

Bacterial Proteins↗
PubMed2005

Coordinated replication and sequestration of oriC and dnaA are required for maintaining controlled once-per-cell-cycle initiation in Escherichia coli.

Escherichia coli cells were constructed in which the dnaA gene was moved to a location opposite oriC on the circular chromosome. In these cells the dnaA gene was replicated with significant delay relative to the origin. Consequently, the period where the newly replicated and hemimethylated oriC was sequestered no longer coincided with the period where the dnaA gene promoter was sequestered. DnaA protein synthesis was therefore expected to continue during origin sequestration. Despite a normal length of the sequestration period in such cells, they had increased origin content and also displayed asynchrony of initiation. This indicated that reinitiation occasionally occurred at some origins within the same cell cycle. The extra initiations took place in spite of a reduction in total DnaA protein concentration to about half of the wild-type level. We propose that this more efficient utilization of DnaA protein results from an increased availability at the end of the origin sequestration period. Therefore, coordinated sequestration of oriC and dnaA is required for maintaining controlled once-per-cell-cycle initiation.

Bacterial Proteins↗
PubMed2004

Host controlled plasmid replication: Escherichia coli minichromosomes.

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.

Chromosomes, Bacterial↗
PubMed2003

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli.

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB-depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.

Amino Acid Sequence↗
PubMed2003

Role of SeqA and Dam in Escherichia coli gene expression: a global/microarray analysis.

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.

Bacterial Outer Membrane Proteins↗
PubMed2003

Stable co-existence of separate replicons in Escherichia coli is dependent on once-per-cell-cycle initiation.

DNA replication in most organisms is regulated such that all chromosomes are replicated once, and only once, per cell cycle. In rapidly growing Escherichia coli, replication of eight identical chromosomes is initiated essentially simultanously, each from the same origin, oriC. Plasmid-borne oriC sequences (minichromosomes) are also initiated in synchrony with the eight chromosomal origins. We demonstrate that specific inactivation of newly formed, hemimethylated origins (sequestration) was required for the stable co-existence of oriC-dependent replicons. Cells in which initiations were not confined to a short interval in the cell cycle (carrying mutations in sequestration or initiation genes or expressing excess initiator protein) could not support stable co-existence of several oriC-dependent replicons. The results show that such stable co-existence of oriC-dependent replicons is dependent on both a period of sequestration that is longer than the initiation interval and a reduction of the initiation potential during the sequestration period. These regulatory requirements are the same as those required to confine initiation of each replicon to once, and only once, per cell cycle.

Cell Cycle↗
PubMed2003

Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division.

In Escherichia coli, the level of the initiator protein DnaA is limiting for initiation of replication at oriC. A high-affinity binding site for DnaA, datA, plays an important role. Here, the effect of extra datA sites was studied. A moderate increase in datA dosage ( approximately fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold. At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected. Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement. In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork. The results suggest that wild-type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function.

Adenosine Triphosphatases↗