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At least 883 records · Page 49Linked to original sources
PubMed2004

The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1beta group without any accessory genes.

The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1beta backbone, which diverged early from the sequenced IncP-1beta plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14.4 kb or 19.0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1beta plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)-tetR-tnpA(IS26) fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D beta-lactamase (bla(NPS-2)); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)-tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1beta plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1beta plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.

Antiporters↗
PubMed1997

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Bacterial Proteins↗
PubMed1994

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

Bacterial Proteins↗
PubMed1976

Complexity of the ultraviolet mutation frequency response curve in Escherichia coli B/r: SOS induction, one-lesion and two-lesion mutagenesis.

Three distinct sections of the ultraviolet mutation frequency response (MFR) curve toward tryptophan prototrophy have been demonstrated in Excherichia coli B/r WP2 trp thy and its uvrA derivative in log-phase growth in minimal medium. The initial section, which appears fluence-squared, may reflect the necessity, if mutation is to result, for induction of two lesions, one located within the potentially mutated genetic locus and the other damaging deoxyribonucleic acid replication and resulting in inducation of the error-prone SOS repair function. A second linear section is ascribed to the continued induction, after exposure above that sufficient for complete SOS expression, of isolated lesions which lead to mutation in potentially mutated loci. The third section demonstrates an increased rate of mutagenesis and suggests the induction of two lesions in proximity which result in additional mutations. Split-exposure studies support the inducible nature of the SOS function and suggest that mutation frequency decline (MFD) is due to exicion resulting from or related to the prevention of SOS induction by inhibition of protein synthesis. Preirradiation tryptophan starvation of the uvr+ strain for 30 min decrease MFR in the first and second sections of the curve. Reduction of MFR in the third section requires more prestarvation time and is blocked by nalidixic acid. The decreased MFR of the first and second sections ascribed to promotion of postirradiation MFD based on excision and that of third section to completion of the chromosome during the prestarvation period.

Bacterial Proteins↗
PubMed1999

Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly.

Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes.

Bacillus subtilis↗
PubMed1971

Division cycle of Myxococcus xanthus. II. Kinetics of stable and unstable ribonucleic acid synthesis.

The kinetics of stable and unstable ribonucleic acid (RNA) synthesis during the division cycle of Myxococcus xanthus growing in a defined medium was determined. Under these conditions, M. xanthus contains one chromosome which is replicated during 80% of the cell cycle. Stable RNA synthesis was measured by pulselabeling an exponential-phase culture with radioactive uridine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of stable RNA synthesis as a function of cell size was determined. Unstable RNA synthesis during the division cycle was determined by correlating the data for stable RNA synthesis with the relative amounts of stable and unstable RNA labeled during the short pulse. The data reported here demonstrate that: (i) cells synthesize both stable and unstable RNA throughout the division cycle; (ii) the rate of stable RNA synthesis increases in two discrete steps, corresponding to average ages of 0.15 and 0.75 generations; (iii) the rate of unstable RNA synthesis exhibits an initial rise, followed by a relatively constant rate of synthesis, and finally, a burst of unstable RNA synthesis prior to septum formation. The half-life of unstable RNA of M. xanthus, generation time of 390 min at 30 C, was 4 min. Comparison of the rates of stable and unstable RNA synthesis indicates noncoordinate RNA synthesis within the normal division cycle.

Autoradiography↗
PubMed1991

Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response.

Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo.

Bacterial Proteins↗
PubMed1992

Genes and their organization in the replication origin region of the bacterial chromosome.

Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.

Amino Acid Sequence↗
PubMed1994

The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids.

The large plasmid, pRmeGR4a, of Rhizobium meliloti GR4 is a nonsymbiotic, self-transmissible replicon that shows a high degree of stability. Plasmid replication and stabilization functions are located on a 4.8-kb PstI fragment. Analysis of the nucleotide sequence of this DNA region revealed the presence of six open reading frames (ORFs) with coding capabilities; all are transcribed in the same direction. Only one of the ORFs (ORF3) seems to be essential for replication. Its predicted protein sequence shows homology with RepC, a protein that has been suggested to be involved in the replication of Agrobacterium Ri and Ti plasmids.

Amino Acid Sequence↗
PubMedDate not supplied

The tus gene of Escherichia coli: autoregulation, analysis of flanking sequences and identification of a complementary system in Salmonella typhimurium.

The tus gene of Escherichia coli encodes a DNA-binding protein that, when bound to terminator sites, blocks replication forks. One of these sites, TerB, is immediately upstream from tus, and we have determined that the 5' end of tus mRNA is in the TerB site, that tus is autoregulated and that pTus is a very low efficiency promoter. Analysis of the DNA upstream from tus and TerB indicates a set of sensor/regulator genes which are comparable to envZ/ompR. Although tus mutants exhibit no growth phenotype in laboratory conditions, Salmonella typhimurium and E. coli have nevertheless maintained similar termination systems. Sequence homology can be demonstrated by Southern hybridizations, and the systems also exhibit functional complementation: the Tus protein of S. typhimurium blocks DNA replication at the TerA site of E. coli.

Bacterial Proteins↗
PubMed1987

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda.

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.

Bacterial Proteins↗
PubMed2001

An analysis of the factory model for chromosome replication and segregation in bacteria.

Recent advances in microscopy have given us important clues as to the nature of chromosome segregation in bacteria. Most current observations favour the view that the process is co-replicational: DNA replication forks are anchored at the cell centre, and the newly replicated DNA is moved towards the cell poles. This scheme can account for orderly segregation even at high growth rates where multiple replication cycles overlap. We argue that there are five distinct activities directly involved in co-replicational segregation dynamics. These we refer to as Push, Direct, Condense, Hold and Clear. We attempt to assign one of these roles to each protein implicated in chromosome segregation. The proposed process is very different from mitosis in eukaryotic cells and perhaps more closely resembles the formation of separate sister chromatids during DNA replication.

Bacteria↗
PubMed1991

A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K.

Understanding the role of Escherichia coli histone-like protein integration host factor (IHF) in replication of R6K plasmid (Dellis, S., and Filutowicz, M. (1991) J. Bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of IHF protein with the plasmid's replication origin (gamma ori). We describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of IHF, in which, on average, a 102-base pair (bp) ori segment is involved. IHF.gamma ori complexes also undergo a two-step conformational change in an IHF concentration-dependent manner when analysed by band shift assay. We believe that the DNA is bent at low IHF concentrations, but folded at high IHF concentrations. This idea is supported by the fact that electrophoretic mobility of the IHF.gamma ori complexes is faster at higher concentrations of IHF. Furthermore, it is shown that the formation of a compact nucleoprotein structure depends on the two regions flanking the AT-rich segment; the iterons to the right and the 106-bp ori domain to the left. Finally we show that IHF protects the entire AT-rich segment of the ori against nuclease cleavage. In addition to the protection, an altered cleavage pattern by DNase I, in the presence of high levels of IHF, was observed within the iterons but not within the 106-bp domain of the ori. Implications of the IHF-mediated gamma ori folding as a possible mechanism protecting the ori from replication inhibition by R6K initiator protein tau are discussed.

Bacterial Proteins↗
PubMed1998

[Function of DnaA protein, the initiator for chromosomal DNA replication in E. coli].

DnaA protein is an initiator for chromosomal DNA replication in E. coli. We have examined the function of the protein to answer the following four questions; 1. How DnaA protein is inactivated after DNA replication for the suppression of re-initiation? 2. How DnaA protein is activated for the initiation of DNA replication? 3. Does DnaA protein have functions other than that for DNA replication? 4. Is DnaA protein is a good target for new antibiotics? In this review, I summarize our recent studies for these questions.

Adenosine Diphosphate↗