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At least 343 records · Page 19Linked to original sources
PubMed1993

Coumermycin A1 inhibits growth and induces relaxation of supercoiled plasmids in Borrelia burgdorferi, the Lyme disease agent.

Coumermycin A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes supercoiling of DNA and is required for bacterial DNA replication. We have investigated the activity of this coumarin antibiotic on Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease. B. burgdorferi was more susceptible than many other eubacteria to coumermycin as well as novobiocin, another coumarin antibiotic; this contrasted with its relative resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic acid, and ciprofloxacin. Coumermycin at 0.2 micrograms/ml inhibited the growth of B. burgdorferi B31 in BSK II medium. A 100-fold-lower concentration induced the relaxation of two negatively supercoiled circular plasmids within 2 h. Plasmid supercoiling was restored within 2 h of removal of coumermycin. These results suggest that B. burgdorferi has a DNA gyrase and that this enzyme's activity is required for growth. Furthermore, structural analogs of coumermycin may be considered as treatments for Lyme disease.

Aminocoumarins↗
PubMed1997

Unraveling a region-specific hyper-recombination phenomenon: genetic control and modalities of terminal recombination in Escherichia coli.

The propensity of the terminus of the Escherichia coli chromosome for recombination has been further explored, using a test based on the selectable loss of a lambda prophage inserted between repeated sequences from Tn10. Terminal recombination appears region-specific and unrelated to replication termination in a strain harboring a major chromosomal rearrangement. It requires RecBC(D) activity and must therefore occur between sister chromosomes, to conserve genomic integrity in spite of DNA degradation by RecBCD. Terminal recombination is maximal in the dif region and its intensity on either side of this recombination site depends on the orientation of the repeated sequences, probably because of the single chi site present in each repeat. Additional observations support the model that the crossover is initiated by single-strand invasion between sister chromosomes followed by RecBCD action as a consequence of DNA breakage due to the initial invasion event. Crossover location within repeats inserted at dif position supports the possibility that sister chromosomes are tightly paired in the centre of the terminal recombination zone. These data reinforce the model that postreplicative reconstruction of nucleoid organization creates a localized synapsis between the termini of sister chromosomes.

Chromosomes, Bacterial↗
PubMed1975

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.

Acetates↗
PubMed1997

The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli.

Despite the widely accepted view that transcription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC. Furthermore, neither gid nor mioC transcription is required in cells deficient in the histone-like proteins Fis or IHF. However, oriC that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of mioC and especially gid is needed to activate oriC only under suboptimal conditions. We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within oriC, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.

Bacterial Proteins↗
PubMed1979

Chromosome replication and cell division in plasmid-containing Escherichia coli B/r.

The kinetics of chromosome replication and cell division have been examined in recA mutants of Escherichia coli B/r containing F' plasmids of various sizes. Plasmid-mediated alterations in growth properties were detected only with the presence of the larger F' plasmids, and were reflected in decreased mean cell sizes and growth rates. The lengths of C and D in all plasmid-containing strains were in accord with the values for plasmid-free parental strains growing with similar generations times. The findings were consistent with an absence of competition between the chromosomal and extrachromosomal replicons for rate-limiting components involved in the initiation of deoxyribonucleic acid synthesis or in the elongation of deoxyribonucleic acid chains.

Cell Division↗
PubMed1983

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome.

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.

Base Sequence↗
PubMed1975

Permanent loss of chromosome initiation in toluene-treated Bacillus subtilis cells.

Initiation of deoxyribonucleic acid replication is absent in Bacillus subtilis cells made permeable by toluene. The absence of initiation may be (i) a temporary removal of toluene, or (ii) irreversibly lost due to damage by toluene treatment to a cellular structure or a process required for chromosome initiation. Washed cells, previously treated with toluene and subsequently washed free of detectable amounts of toluene, have the same characteristics of toluene-treated cells in which toluene is not removed. The continued absence of initiation in the washed cells indicates a permanent loss of initiation in cells treated with toluene. Protein synthesis is also inhibited irreversibly by toluene treatment, indicating damage to translation as a possible mechanism for loss of initiation of toluene-treated cells.

Bacillus subtilis↗
PubMed2001

Polarization of the Escherichia coli chromosome. A view from the terminus.

The E. coli chromosome replication arms are polarized by motifs such as RRNAGGGS oligomers, found preferentially on leading strands. Their skew increases regularly from the origin to dif (the site in the center of the terminus where chromosome dimer resolution occurs), to reach a value of 90% near dif. Convergent information indicates that polarization in opposite directions from the dif region controls tightly the activity of dif, probably by orienting mobilization of the terminus at cell division. Another example of polarization is the presence, in the region peripheral to the terminus, of small non-divisible zones whose inversion interferes with spatial separation of sister nucleoids. The two phenomena may contribute to the organization of the Ter macrodomain.

Amino Acid Motifs↗
PubMed1970

Control of deoxyribonucleic acid and ribonucleic acid synthesis in pyrimidine-limited Escherichia coli.

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.

Autoradiography↗
PubMed1979

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.

Bacterial Proteins↗
PubMed1974

Control of deoxyribonucleic acid synthesis in a Bacillus subtilis mutant temperature sensitive for initiation of chromosome replication.

We used a Bacillus subtilis mutant described previously, which is temperature sensitive for initiation of replication. The inhibition of deoxyribonucleic acid synthesis occurring at 45 C was shown to be reversed when the temperature is lowered even in the absence of protein synthesis. If the bacteria are returned to 30 C, after a prior period at 45 C, they are able to initiate the first round of replication in the presence of chloramphenicol, but the initiation of the second round still requires protein synthesis. This paper shows that the proteins necessary to initiate the second round of replication can be present in bacteria long before this round is initiated. In addition, the appearance of these proteins seems to be influenced by the length of the previous 45 C period. Although similar reinitiation kinetics are observed at 30 C after prior 45 C periods of 30 or 65 min, the ability to initiate the second round without further protein synthesis appears much earlier after a longer exposure at 45 C. To explain these results, a hypothesis is presented which assumes that two different proteins are both necessary for initiation. Only one of these proteins could be accumulated at 45 C during the inhibition of deoxyribonucleic acid synthesis. A peculiarity of initiation material in mutant Ts 37 is that it may be active at 45 C if it has been exposed previously at 30 C.

Bacillus subtilis↗
PubMed1988

Design, synthesis, and properties of (4S)-7-(4-amino-2-substituted-pyrrolidin-1-yl)quinolone-3-carboxylic acids.

The quinolonecarboxylic acids constitute a class of extremely potent and orally active broad-spectrum antibacterial agents. These compounds have been shown to inhibit DNA gyrase, a key enzyme in bacterial DNA replication. The 7-(3-aminopyrrolidinyl)quinolone A-60969 (1) is a particularly potent member of this class and is currently undergoing clinical evaluation. We have studied a series of enantiomerically homogeneous (4S)-7-(4-amino-2-substituted-pyrrolidinyl)quinolones in an effort to utilize the 2-position of the pyrrolidine moiety to improve upon the solubility and pharmacokinetic properties of this class of compounds while still maintaining potent antibacterial activity. We have found that the absolute stereochemistry at the 2-position of the pyrrolidine ring is critical to the maintenance of such activity. In this paper, we report the full details of the asymmetric synthesis and the in vitro and in vivo structure-activity relationships of this series of compounds as well as the physiochemical properties, such as water solubility and log P, associated with the structural modifications. We also discuss the pharmacokinetic properties of several of these compounds in mice and the pharmacokinetics of 59, which has the best overall properties of agents in this study, in dog.

Animals↗
PubMed1987

The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation.

The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome. The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain. This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC. The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned. The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division. B. subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC. This manipulation did not introduce any readily detectable auxotrophic requirement. Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.

Bacillus subtilis↗
PubMed2003

Gene essentiality determines chromosome organisation in bacteria.

In Escherichia coli and Bacillus subtilis, essentiality, not expressivity, drives the distribution of genes between the two replicating strands. Although essential genes tend to be coded in the leading replicating strand, the underlying selective constraints and the evolutionary extent of these findings have still not been subject to comparative studies. Here, we extend our previous analysis to the genomes of low G + C firmicutes and gamma-proteobacteria, and in a second step to all sequenced bacterial genomes. The inference of essentiality by homology allows us to show that essential genes are much more frequent in the leading strand than other genes, even when compared with non- essential highly expressed genes. Smaller biases were found in the genomes of obligatory intracellular bacteria, for which the assignment of essentiality by homology from fast growing free-living bacteria is most problematic. Cross-comparisons used to assess potential errors in the assignment of essentiality by homology revealed that, in most cases, variations in the assignment criteria have little influence on the overall results. Essential genes tend to be more conserved in the leading strand than average genes, which is consistent with selection for this positioning and may impose a strong constraint on chromosomal rearrangements. These results indicate that essentiality plays a fundamental role in the distribution of genes in most bacterial genomes.

Bacillus subtilis↗
PubMed1992

IciA protein, a specific inhibitor of initiation of Escherichia coli chromosomal replication.

Specific binding of IciA protein to the 13-mers in the origin of a minichromosome (oriC) inhibits initiation of replication in vitro by blocking the opening of this region effected by the initiator DnaA protein (Hwang, D.S., and Kornberg, A. (1990) Cell 63, 325-331). Isolation of the iciA gene (Thöny, B., Hwang, D.S., Fradkin, L., and Kornberg, A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4066-4070) has made possible the construction of an IciA-overproducing strain, which in turn has simplified the isolation of a large quantity of the protein, indistinguishable from that of the wild-type strain. Based on sedimentation and gel filtration, the IciA protein is an elongated dimer of a 33.4-kDa subunit. The specific binding of IciA protein to the 13-mers was stable for 2 h at 30 degrees C. The amounts of IciA protein, detected by immunoassays, increased 4-fold compared with levels (about 100 dimers) in log-phase cells whereas levels of DnaA protein decreased upon entry of cells into the stationary phase.

Bacterial Proteins↗
PubMed1998

Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein.

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.

Bacterial Outer Membrane Proteins↗
PubMed2005

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus.

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.

Bacterial Proteins↗
PubMed2002

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins.

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long-terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5' ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Streptomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their cosegregation and co-evolution. All the Tpg polypeptides are similar in length (184-185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA-binding 'thumb' domain of HIV reverse transcriptase, and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or the proposed membrane binding.

Amino Acid Sequence↗