Biomedical subjects

F G Hansen

Publications and source records attributed to F G Hansen.

At least 19 recordsLinked to original sources

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↗

PubMed1991

Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium.

The DnaA protein concentration was determined in five different Escherichia coli strains and in Salmonella typhimurium LT2 growing at different growth rates. The DnaA protein concentration was found to be invariant over a wide range of growth rates in the four E. coli K-12 strains and in S. typhimurium. In E. coli B/r the DnaA protein concentration was generally higher than in the K-12 strains, and it increased with decreasing growth rates. For all the strains, there appears to be a correlation between the DnaA protein concentration and the initiation mass. This supports the concept of the concentration of DnaA protein setting the initiation mass and, thus, that the DnaA protein is a key molecule in the regulation of initiation of chromosome replication in members of the family Enterobacteriaceae.

Bacterial Proteins↗

PubMed1991

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.

Aspartic Acid↗

PubMed1989

The DnaA protein determines the initiation mass of Escherichia coli K-12.

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

Bacterial Proteins↗

PubMed1989

Cloning and characterization of the Escherichia coli phosphoglycerate kinase (pgk) gene.

The pgk gene of Escherichia coli coding for the phosphoglycerate kinase was subcloned from the Carbon and Clarke collection plasmid pLC33-5. The position and direction of transcription of the pgk gene was determined by Tn5 insertion mutagenesis. Analysis of proteins encoded from these plasmids showed that the pgk gene product is a 40-kDa protein, and that the gene is transcribed from two promoters, one immediately in front of the gene and one in front of an upstream gene coding for a 38-kDa polypeptide of unknown function. The position of the Pgk protein on two-dimensional O'Farrel gels was identified, and from this we conclude that it is one of the proteins induced by anaerobiosis [Smith and Neidhardt, J. Bacteriol. 154 (1987) 336-343]. The pgk gene was also found to show growth phase regulation; the synthesis of Pgk protein was induced more than ten-fold during transition from the exponential to the stationary growth phase.

Anaerobiosis↗

PubMed1989

In vivo studies of DnaA binding to the origin of replication of Escherichia coli.

We have shown that DnaA, a protein required for initiation of DNA replication in Escherichia coli, binds to three of four DnaA binding sequences in the replicative origin oriC (boxes R1, R2 and R4). Protein-oriC DNA interactions in minichromosomes carried by wild-type and dnaA mutant strains were demonstrated by in vivo footprinting using dimethylsulfate treatment of intact cells. The same characteristic enhancement/protection pattern was seen in wild-type minichromosomes or mutants defective in oriC function but carrying the four DnaA boxes. Minichromosomes in dnaA (Ts) mutants showed no protein binding at non-permissive temperatures and reduced binding even at permissive temperatures. In vivo footprints of the wild-type strain were identical to those obtained in vitro using purified DnaA proteins and oriC DNA. Transcription into oriC affected the binding of DnaA protein to the DnaA boxes. These findings suggest that the protein causing the in vivo footprints at oriC is DnaA.

Bacterial Proteins↗

PubMed1989

Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli.

A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth. The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way. The appY gene was mapped to 13 min on the E. coli genetic map. The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined. The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators.

Amino Acid Sequence↗