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At least 595 records · Page 33Linked to original sources
PubMed1987

Initiation of plasmid pC194 replication and its control in Bacillus subtilis.

By deletion analysis we have defined a 1.1 kb segment required for driving autonomous replication of the plasmid pC194. The minimal replicon specifies a positive, RepH, and a negative, Inc8A, trans-acting product and their target sites. The RepH product has a Mr of 34.1 kDa, could be overproduced, and binds specifically to the pC194 origin region. By trans complementation studies we have shown that pC194 replication is indirectly controlled at the level of RepH synthesis by a negative product, IncA, that is transcribed within the repH transcription unit in the opposite direction ("antisense RNA"). The antisense RNA regulates the RepH synthesis by a mechanism that seems to involve RNA/RNA interaction in a manner that interferes with translation. In addition, an autoregulatory control might be operative.

Bacillus subtilis↗
PubMed2003

Strategies for helicase recruitment and loading in bacteria.

DNA replication initiation in prokaryotes and eukaryotes requires the recruitment and loading of a helicase at the replication origin. To subsequently unwind the double-stranded DNA, the helicase must be properly positioned on the separated DNA strands. Several studies have revealed similarities and differences in the mechanisms used by different autonomously replicating DNA elements (replicons) for recruitment and activation of the appropriate helicase. Of particular interest are plasmid replicons that are adapted for replication in diverse bacterial hosts and are therefore intriguingly able to exploit the helicases of distantly related bacterial species. The different molecular mechanisms by which replicons recruit and load helicases are only just beginning to be understood.

Bacterial Proteins↗
PubMed2004

Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6.

Pseudomonas sp. strain ND6, which was isolated from industrial wastewater in Tianjin, China, was capable of dissimilating naphthalene as sole carbon and energy sources. We identified one plasmid, pND6-1, which was associated with the metabolism of naphthalene and determined the complete nucleotide sequence of pND6-1 (101,858 bp) using a whole-genome-shotgun approach. Computational analyses indicated that the naphthalene metabolism of the strain ND6 is associated with this plasmid. This is the first report of a complete sequence of naphthalene catabolic plasmid. pND6-1 encodes 102 putative coding sequences (CDSs). Among them, 23 CDSs were predicted to be involved in naphthalene catabolism, 14 were predicted to be involved in transposition and integration, 2 encoded putative transporters, 3 were putative transcriptional regulators, and 9 were proteins necessary for plasmid replication and partitioning. Most of the naphthalene catabolic genes of pND6-1 have 99-100% identity in amino acid sequences homologous to their nearest counterparts found in plasmid pDTG1, NAH7 and in a chromosome region in Pseudomonas stutzeri AN10 except for two duplicated genes (ND013 and ND016). Results of this study indicated that globally distributed naphthalene catabolic genes are highly conserved among different bacterial species.

Bacterial Proteins↗
PubMed1993

Methylated cytosine at Dcm (CCATGG) sites in Escherichia coli: possible function and evolutionary implications.

The frequency and distribution of methylated cytosine (5-MeC) at CCATGG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.

Amino Acids↗
PubMed1992

Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.

Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.

Alkylation↗
PubMed1997

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Bacillus subtilis↗
PubMed2002

Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins.

RelE and ChpAK (MazF) toxins of Escherichia coli have previously been described as proteins that mediate efficient cell killing. We show here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate. Later induction of transcription of the antitoxin genes relB or chpAI fully reversed the static condition induced by RelE and ChpAK respectively. We also provide a mechanistic explanation for these findings. Thus, induction of relE transcription severely inhibited translation, whereas induction of chpAK transcription inhibited both translation and replication. Hence, most likely, lack of colony formation is due to inhibition of translation in the case of relE and inhibition of translation and/or replication in the case of chpAK. Consistent with this proposal, later induction of transcription of the cognate antitoxin genes simultaneously reversed cell stasis and the inhibitory effects of RelE and ChpAK on macromolecular syntheses. These results preclude that RelE and ChpAK mediate cell killing during the conditions used here. In vivo and in vitro analyses of a mutant RelE protein supported that inhibition of colony formation was due to inhibition of translation.

Bacterial Toxins↗
PubMed1986

Positive and negative roles of an initiator protein at an origin of replication.

The properties of mutants in the pir gene of plasmid R6K have suggested that the pi protein plays a dual role; it is required for replication to occur and also plays a role in the negative control of the plasmid copy number. In our present study, we have found that the pi level in cell extracts of Escherichia coli strains containing R6K derivatives is surprisingly high (approximately equal to 10(4) dimers per cell) and that this level is not altered in cells carrying high copy number pir mutants. The wild-type and a high copy mutant (Cos405) pir gene were inserted downstream of promoters of different strengths to measure the copy number of an R6K gamma replicon as a function of a 1000-fold range of intracellular pi concentrations. The data demonstrate that reducing the intracellular level of pi to 5% of its normal value can result in a substantial increase in copy number of a gamma origin replicon and that a pi level less than 1% of normal is still permissive for replication. Conversely, increasing the pi level even a few-fold above normal results in a marked inhibition of replication of plasmids containing a single, two, or all three of the R6K origins (alpha, beta, and gamma). We have also shown that the replication inhibition mediated by excess pi is greatly reduced by the pir405 Cos mutation. These results demonstrate that the total level of pi protein is not rate-limiting for a gamma replicon. We have also determined the sensitivity of the pir gene promoter to a wide range of pi concentrations. The activity of this promoter is stimulated by very low pi levels and is almost entirely inhibited when the protein is overproduced 2-fold.

Bacterial Proteins↗
PubMed1991

A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication.

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.

Bacillus subtilis↗
PubMed1996

Escherichia coli strains in which chromosome replication is controlled by a P1 or F replicon integrated into oriC.

We report the construction of intP1 and intFs strains, in which the basic replicon from either plasmid P1 or plasmid F (oriS) has been integrated in both orientations into the origin of replication, oriC, of the Escherichia coli chromosome. In these strains, oriC is no longer functional and chromosome-replication is instead controlled by the integrated plasmid replicon. The strains were viable, showing that the deviation from normal chromosome-replication control was not large enough to prohibit cell survival. The strains showed a broader cell-size distribution than a wild-type strain and were more filamentous in rich than in minimal media, although cells of wild-type size were also present. Cells which contained aberrantly shaped or aberrantly distributed nucleoids were also observed. Marker-frequency analysis indicated that chromosome replication was predominantly bidirectional in both intFs strains. In the intP1 strains, the degree of bidirectionality depended upon the orientation of the integrated replicon.

Chromosomes, Bacterial↗
PubMed1999

Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication.

The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication.

Bacterial Proteins↗
PubMed2002

Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372.

The DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. Acidic phospholipids decrease its affinity for adenine nucleotides, and re-activate the ADP-bound form to the ATP-bound form. We have previously reported that two mutant forms, DnaAR328E and DnaAK372E, have decreased affinity for cardiolipin (CL). In the present study, we constructed a mutant DnaA protein, DnaA435, with both R328E and K372E, and compared its biochemical characteristics with those of DnaAR328E and DnaAK372E. DnaA435 could bind to oriC DNA, but did not bind ATP or ADP. In DnaA435, compared with DnaAR328E and DnaAK372E, CL caused less inhibition of oriC DNA binding, suggesting that amino acids R328 and K372 are involved in the interaction of DnaA with acidic phospholipids. DnaA435 could initiate DNA synthesis on oriC both in vivo and in vitro. Based on these results, we propose that ATP activates DnaA protein by changing its higher order structure around R328 and K372.

Adenosine Diphosphate↗
PubMed1971

Quantitative aspects of deoxyribonucleic acid renaturation: base composition, state of chromosome replication, and polynucleotide homologies.

The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was established that high guanosine plus cytosine (GC) DNA renatures faster than expected from analytical measurement of its molecular weight. A calculated correction factor of 1.8% of the observed C(0)t(.5) is required for every mole per cent GC difference from 51% GC. The correction factor is now established in the range of 32 to 65% GC. Renaturation of DNA mixtures prepared from pairs of organisms has been studied. When no similarity existed between the two organisms, the observed C(0)t(.5) of the mixture was the sum of the independently determined C(0)t(.5) values. Lack of additivity was correlated with similarities in polynucleotide sequence of the reassociating DNA molecules. A quantitative relationship was formulated to relate C(0)t(.5) values of renatured DNA mixtures to per cent binding ("homology"). Finally, it was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.

Autoradiography↗
PubMed2000

Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E. coli.

The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis-E. coli shuttle plasmid was designed to allow the insertion of either the Terl (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP-Terl complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex. The specificity effect is of less significance for an RTP-Ter complex functioning in B. subtilis.

Bacillus subtilis↗
PubMed1981

Conditional lethality of Escherichia coli strains carrying dnaE and dnaQ mutations.

A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at 28 degrees C but not at 30 degrees C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. A dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperature-sensitive growth mutations were combined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.

Alleles↗
PubMed1991

Mutations in the Escherichia coli dnaG gene suggest coupling between DNA replication and chromosome partitioning.

Eleven conditional lethal dnaG(Ts) mutations were located by chemical cleavage of heteroduplexes formed between polymerase chain reaction-amplified DNAs from wild-type and mutant dnaG genes. This entailed end labeling one DNA strand of the heteroduplex, chemically modifying the strands with hydroxylamine or osmium tetroxide (OsO4) at the site of mismatch, and cleaving them with piperidine. The cleavage products were electrophoresed, and the size corresponded to the position of the mutation with respect to the labeled primer. Exact base pair changes were then determined by DNA sequence analysis. The dnaG3, dnaG308, and dnaG399 mutations map within 135 nucleotides of one another near the middle of dnaG. The "parB" allele of dnaG is 36 bp from the 3' end of dnaG and 9 bp downstream of dnaG2903; both appear to result in abnormal chromosome partitioning and diffuse nucleoid staining. A suppressor of the dnaG2903 allele (sdgA5) maps within the terminator T1 just 5' to the dnaG gene. Isogenic strains that carried dnaG2903 and did or did not carry the sdgA5 suppressor were analyzed by a combination of phase-contrast and fluorescence microscopy with 4',6-diamidino-2-phenylindole to stain DNA and visualize the partitioning chromosome. Overexpression of the mutant dnaG allele corrected the abnormal diffuse-nucleoid-staining phenotype associated with normally expressed dnaG2903. The mutations within the dnaG gene appear to cluster into two regions which may represent distinct functional domains within the primase protein.

Bacterial Proteins↗
PubMed2001

Ongoing evolution of strand composition in bacterial genomes.

We tried to identify the substitutions involved in the establishment of replication strand bias, which has been recognized as an important evolutionary factor in the evolution of bacterial genomes. First, we analyzed the composition asymmetry of 28 complete bacterial genomes and used it to test the possibility that asymmetric deamination of cytosine might be at the origin of the bias. The model showed significant correlation to the data but left unexplained a significant portion of the variance and indicated a systematic underestimation of GC skews in comparison with TA skews. Second, we analyzed the substitutions acting on the genes from five fully sequenced Chlamydia genomes that had not suffered strand switch since speciation. This analysis showed that substitutions were not at equilibrium in Chlamydia trachomatis or in C. muridarum and that strand bias is still an on-going process in these genes. Third, we identified substitutions involved in the adaptation of genes that had switched strands after speciation. These genes adapted quickly to the skewed composition of the new strand, mostly due to C-->T, A-->G, and C-->G asymmetric substitutions. This observation was reinforced by the analysis of genes that switched strands after divergence between Bacillus subtilis and B. halodurans. Finally, we propose a more extended model based on the analysis of the substitution asymmetries of CHLAMYDIA: This model fits well with the data provided by bacterial genomes presenting strong strand bias.

Bacteria↗