Tove Atlung

H‐NS: a modulator of environmentally regulated gene expression

H‐NS is a small chromatin‐associated protein found in enterobacteria. H‐NS has affinity for all types of nucleic acids but binds preferentially to intrinsically curved DNA. The major role of H‐NS is to modulate the expression of a large number of genes, mostly by negatively affecting transcription. Many of the H‐NS‐modulated genes are regulated by environmental signals, and expression of most of these genes is positively regulated by specific transcription factors. Therefore one of the purposes of H‐NS could be to repress expression of some genes under conditions characteristic of a non‐intestinal environment, but allow expression of specific genes in response to certain stimuli in the intestinal environment. The hns gene is autoregulated. In vivo the H‐NS to DNA ratio is fairly constant except during cold shock, when it increases three‐ to fourfold. In this review we propose that only the preferential binding to intrinsically curved DNA plays a role under normal growth conditions, and we discuss the different mechanisms by which H‐NS might affect gene expression and how H‐NS could be involved in the response to different stress situations. Finally, we summarize the evolutionary and functional relationship between H‐NS and the homologous StpA.

The initiator titration model: computer simulation of chromosome and minichromosome control.

The initiator titration model was formulated to explain the initiation control of the bacterial chromosome. In particular, features concerning the replication behaviour of minichromosomes, such as their high copy number and Escherichia colis ability to coinitiate chromosome and many minichromosome origins, were considered during the formulation of the model. The model is based on the initiator protein DnaA and its binding sites, DnaA boxes, in oriC, in the dnaA promoter and at other positions on the chromosome. Another important factor in the model is the eclipse period created by the hemimethylation of a new oriC which makes it refractory to initiation. The model was analysed by computer simulations using a stochastic approach varying the different input parameters, and the resulting computer cells were compared with data on living E. coli cells. Here we present the outcome of a few of these simulations concerning the eclipse period, in silico-shift experiments blocking initiation or elongation of replication, and introduction of minichromosomes into the computer cells. We also discuss the synthesis of DnaA protein in the computer cells. From our simulations, we conclude that, whether true or not, the model can mimic the in vivo initiation control of E. coli.

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

SummaryIncreased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible λpL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.

Physical mapping and nucleotide sequence of the rnpA gene that encodes the protein component of ribonuclease P in Escherichia coli

The rnpA gene, coding for the protein component of ribonuclease P (RNase P), was allocated to the dnaA region at 83 min of the E. coli K-12 map. This was accomplished through analysis of recombinant pBR322 plasmids, some of which complemented the temperature sensitivity of a strain carrying the rnpA 49 allele and restored the RNA processing activity. Although the temperature sensitivity of a strain carrying the rnp-241 allele could not be complemented by the rnpA+ plasmid, the RNA-processing activity was restored, suggesting that the rnp-241 mutation is allelic with rnpA 49. In this analysis we also found two genes coding for proteins (60 and 50 kDal) of unknown function. The order of the genes located in this region is in the clockwise orientation: rpmH (5.4 kDal; ribosomal protein L34), rnpA (14 kDal; protein component of RNase P), a gene for a 60-kDal protein (inner membrane protein), a gene for a 50-kDal protein, and tnaA. All these genes are expressed in the clockwise orientation. From the DNA sequence of the rnpA gene region a very basic polypeptide with an Mr of 13773 could be deduced. We conclude that this polypeptide is the rnpA gene product, and is the protein component of RNase P. Comparison with previously published data on the transcription of rpmH suggests that the rnpA gene is the second gene in the rpmH operon.

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.

The nucleotide sequence of the dnaA gene promoter and of the adjacent rpmH gene, coding for the ribosomal protein L34, of Escherichia coli.

The nucleotide sequence was determined of a 945‐bp EcoRI fragment from the Escherichia coli K‐12 chromosome at 82 min containing the promoter region of the dnaA gene. This nucleotide sequence contained a coding sequence identical to the amino acid sequence of the ribosomal protein L34, designated rpmH . The rimA mutation, which affects the maturation of 50S ribosomal particles, may be an allele of the rpmH gene since it maps close to, or within, the L34 coding sequence. The rpmH gene and the dnaA gene are transcribed in the clockwise and counter‐clockwise direction, respectively. Nuclease S1 mapping of transcripts indicated the existence of two major promoters for the L34 gene and two promoters for the dnaA gene within the 945‐bp EcoRI fragment.

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

SummaryFlow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the λpL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter λpL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

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

SummaryPlasmids 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.

A versatile method for integration of genes and gene fusions into the λ attachment site of Escherichia coli

Abstract We have developed a versatile method for integration of modified genes and gene fusions into the bacteriophage λ attachment site (attB) of the Escherichia coli chromosome. The method relies on two components: (7) a DNA integration cassette, flanked by multiple restriction enzyme sites, which contains the λ attP site and, as a selectable marker, the Tn5 aphA gene conferring kanamycin resistance (KmR); and (2) a plasmid with the λ int gene transcribed from the tet promoter. A fragment carrying the gene in question is ligated to the integration cassette, resulting in a circular piece of DNA unable to replicate. The ligation product is then transformed into a strain that contains the int-carrying plasmid. Selection for KmR results in colonies with the cassette integrated into the attB site of the E. coli chromosome. This method was used for integration of several lacZ and phoA promoter fusions. The integration products were analyzed by Southern hybridization. In addition, we found, fortuitously, that the ligated DNA circles could also integrate by homologous recombination, although usually at a much lower frequency than the Int-mediated integration into attB.

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.