Biomedical subjects

F G Hansen

Publications and source records attributed to F G Hansen.

At least 19 recordsLinked to original sources

PubMed2002

Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli.

Flow cytometric analysis showed that the hns205 and hns206 mutants, lacking the abundant nucleoid-associated protein H-NS, have decreased origin concentration, as well as a low number of origins per cell (ploidy). The most striking observation was that the low ploidy was due to a very short replication time, e.g., at 30 degrees C it was halved compared to that of the hns(+) strain. The decreased origin concentration was not caused by a decreased dnaA gene expression, and the hns206 mutant had normal DnaA protein concentrations. The replication phenotypes of the hns206 mutant were independent of RpoS. Cells overproducing H-NS from a LacI-controlled plasmid had a normal origin concentration, indicating that H-NS is not controlling initiation. A wild-type H-NS concentration is, however, required to obtain a wild-type origin concentration, since cells with an intermediate H-NS concentration had an intermediate origin concentration. Two lines of evidence point to an indirect effect of H-NS on initiation. First, H-NS did not show high-affinity binding to any part of oriC, and H-NS had no effect on transcription entering oriC from the mioC promoter. Second, in a shift experiment with the hns206 mutant, when H-NS protein was induced to wild-type levels within 10 min, it took more than one generation before the origin concentration started to increase.

Bacterial Proteins↗

PubMed2001

Characterisation of the allelic variation in the rpoS gene in thirteen K12 and six other non-pathogenic Escherichia coli strains.

The nucleotide sequence of rpoS, the gene for the stress sigma factor, was determined in 13 different K12 strains of Escherichia coli. The results indicate that the original K12 isolate carried an amber mutation at codon 33, which in 50% of the derivatives is mutated by a single base substitution to a coding triplet, in most cases to CAG encoding glutamine. The six non-K12 strains examined here had GAG, encoding glutamate, in position 33. The two most divergent strains had three and seven neutral substitutions in rpoS and carried insertions of 2100 and 2900 bp, respectively, just downstream of the gene. The genetic variations in rpoS were compared with the variation in RpoS-related phenotypes, by measuring catalase (KatE) activity, glycogen accumulation and acid phosphatase levels, and a katEp-gfp fusion was used to visualise katE gene transcription. The RpoS phenotypes of the six rpoS(33E) strains varied significantly more than that of the K12 rpoS(33Q) strains, especially with respect to acid phosphatase levels. This was due to the absence of the gene for the transcriptional activator AppY from four of the rpoS(33E) strains, while all the K12 derivatives carried this gene. When cloned into a LacI-controlled vector and compared in a rpoS::Tn 10 background, the RpoS(33Q) and RpoS(33E) variants showed the same activity.

Alleles↗

PubMed2001

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Crystallography, X-Ray↗

PubMed2000

The eclipse period of Escherichia coli.

The minimal time between successive initiations on the same origin (the eclipse) in Escherichia coli was determined to be approximately 25-30 min. An inverse relationship was found between the length of the eclipse and the amount of Dam methyltransferase in the cell, indicating that the eclipse corresponds to the period of origin hemimethylation. The SeqA protein was absolutely required for the eclipse, and DnaA titration studies suggested that the SeqA protein prevented the binding of multiple DnaA molecules on oriC (initial complex formation). No correlation between the amount of SeqA and eclipse length was revealed, but increased SeqA levels affected chromosome partitioning and/or cell division. This was corroborated further by an aberrant nucleoid distribution in SeqA-deficient cells. We suggest that the SeqA protein's role in maintaining the eclipse is tied to a function in chromosome organization.

Bacterial Outer Membrane Proteins↗

PubMed2000

Two types of cold sensitivity associated with the A184-->V change in the DnaA protein.

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.

Alanine↗

PubMed2000

Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants.

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

Bacterial Proteins↗

PubMed1999

Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12.

The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold. The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184. In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid. The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells.

Bacterial Proteins↗

PubMed1999

Low-temperature-induced DnaA protein synthesis does not change initiation mass in Escherichia coli K-12.

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.

Amino Acid Sequence↗

PubMed1999

DnaA boxes are important elements in setting the initiation mass of Escherichia coli.

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.

Bacterial Proteins↗

PubMed1996

The initiation mess?

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the 'initiation mass'. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co-operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective preinitiation DnaA-oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli.

Bacterial Proteins↗

PubMed1995

Reinitiation kinetics in eight dnaA(Ts) mutants of Escherichia coli: rifampicin-resistant initiation of chromosome replication.

The kinetics of reinitiation of chromosome replication of eight dnaA(Ts) mutants was investigated in an isogenic set of strains. Five mutants (167, 46, 601, 606 and 5) are classified as reversible, since they can reinitiate at 30 degrees C without protein synthesis, whereas the other three (508, 205, 204) require protein synthesis. In the presence of protein synthesis, reversible mutants initiate one round of replication rapidly after a shift to 30 degrees C, indicating that they contain active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15-20 min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42 degrees C and only require synthesis of small amounts of new DnaA protein before initiation at 30 degrees C, whereas dnaA205 accumulates DnaA protein for some time at 30 degrees C before reaching the initiation threshold. Three of the reversible mutants (5, 601, and 606) exhibited, in addition to the protein synthesis-independent initiation capacity, an RNA synthesis-independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein, strongly suggesting that mutant DnaA protein is responsible for both.

Bacterial Proteins↗

PubMed1995

Reversibility of DnaA protein activity in the 'irreversible' dnaA204 mutant of Escherichia coli.

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature down-shift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42 degrees C. The data indicate that the dnaA204 mutant after a shift to 42 degrees C still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30 degrees C, and after induction of DnaA protein synthesis at 42 degrees C. A model describing the processing of DnaA protein in mutants and in the wild type is presented.

Bacterial Proteins↗

PubMed1995

Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(nuII) rnh mutant of Escherichia coli.

The kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(nuII) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild-type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild-type concentration (24 ng of DnaA protein per ml cells at OD450 = 1.0). At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30 min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.

Bacterial Proteins↗

PubMed1994

The initiation cascade for chromosome replication in wild-type and Dam methyltransferase deficient Escherichia coli cells.

'Newborn' Escherichia coli B/r cells, obtained by membrane elution, were used to study the cell cycles of wild-type and Dam methyltransferase mutants. In wild-type cells, initiation of chromosome replication was synchronous and tightly controlled. In dam mutants, initiation was altered, but not random. We propose that this is due to the absence of an initiation cascade caused by liberated DnaA molecules, and that this cascade normally synchronizes initiation. The dam- cells contained mainly two, three or four replication origins, and this affected nucleoid partitioning as well as cell division. In cultures growing with a 50 min doubling time, a variety of cell cycles were present and half the origins were used every 25 min. Some cells had a 25 min interdivision time, whereas others had an interdivision time longer than the generation time. Partitioning of nucleoids containing unequal numbers of replication origins could also be readily observed by fluorescence microscopy in the dam mutant. Based upon these observations we propose that the dam mutant is also an initiation cascade mutant.

Bacterial Proteins↗

PubMed1994

Complete nucleotide sequence of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3 and its correlation with biological properties.

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

Amino Acid Sequence↗

PubMed1993

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli.

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.

Bacterial Proteins↗

PubMed1992

Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli.

Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.

Alanine↗

PubMed1992

Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome.

Cloning of an autonomously replicating sequence (ars) from the origin region of Bacillus subtilis was previously unsuccessful because of the strong incompatibility exerted by sequences located within the oriC region. Using an ars searching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of the oriC region, we have succeeded in isolating ars fragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA-box regions (non-translatable regions containing multiple repeats of DnaA-box) separated by the dnaA gene. Neither one of the DnaA-box regions by itself showed ars activity. When constructed as oriC plasmids, the dnaA coding region could be removed without affecting ars activity. The minimum distance between the two DnaA-box regions obtained so far is 274 bp. The copy number of the oriC plasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into chromosome.

Amino Acid Sequence↗