RESUMEN
We recently proposed that guillotining of dimer chromosomes occurs at cell division in resolvase mutants of Escherichia coli. This was based on the abnormal pattern of cell division observed in 10-14% of the cells in microcolonies of xerC, xerD and dif mutants. A prediction of this guillotining is that DNA degradation should occur in the terminus region, in the vicinity of the dif locus. We have tested this by DNA-DNA hybridization and have observed that dif was absent in about 22% of the chromosomes in exponentially growing xerC mutants. A locus 206 kb from dif was not affected by this degradation. We have also observed that degradation did not occur in xerC recD mutants, and that the low efficiency of plating associated with the Dif phenotype was suppressed in this strain. A model is proposed in which rapid degradation of the terminus region does not occur in recD mutants following guillotining, and that this permits the initiation of repair of broken dimer chromosomes prior to completion of cell division.
Asunto(s)
Replicación del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Transposasas/genética , División Celular , Mapeo Cromosómico , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Fenotipo , Recombinasas , Recombinación GenéticaRESUMEN
We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA-negative cells. If SOS induction was blocked by lexA3 (Ind-), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/crecimiento & desarrollo , Integrasas , Respuesta SOS en Genética/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular , Cromosomas Bacterianos , ADN Nucleotidiltransferasas/genética , Dimerización , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes , Indoles , Mutagénesis , Fenotipo , RecombinasasRESUMEN
The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.
Asunto(s)
Antiinfecciosos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Norfloxacino/farmacología , Transposasas/efectos de los fármacos , Transposasas/genética , Girasa de ADN , Topoisomerasa de ADN IV , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli , Proteínas de la Membrana/genética , Mutación , Recombinasas , Transposasas/metabolismoRESUMEN
Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site-specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition.
Asunto(s)
Cromosomas Bacterianos , Proteínas de la Membrana/metabolismo , Sitios de Unión , Citoplasma/metabolismo , Dimerización , Proteínas de Escherichia coli , Proteínas de la Membrana/genética , Recombinación GenéticaRESUMEN
Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Recombinación Genética , Intercambio de Cromátides Hermanas/genética , Transposasas/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Cromosomas Bacterianos/genética , Medios de Cultivo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/crecimiento & desarrollo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/metabolismo , Genes Bacterianos , Mutación , RecombinasasRESUMEN
The dif locus is a RecA-independent resolvase site in the terminus region of the chromosome of Escherichia coli. The locus reduces dimer chromosomes, which result from sister chromatid exchange, to monomers. A density label assay demonstrates that recombination occurs at dif, and that it requires XerC and XerD. The frequency of this recombination is approximately 14% per site per generation, which is doubled in polA12 mutants. We have determined that recombination occurs late in the cell cycle, and that resolution is blocked if cell division is inhibited with cephalexin or by a ftsZts mutation. Fluorescence microscopy has demonstrated that abnormal nucleoids are present in cells incubated in cephalexin, and this is increased in polA12 mutants.
Asunto(s)
Cromosomas Bacterianos , Proteínas de Escherichia coli , Escherichia coli/genética , Integrasas , Sitios de Unión , División Celular , Cromosomas Bacterianos/química , Cromosomas Bacterianos/fisiología , ADN Nucleotidiltransferasas/genética , Escherichia coli/citología , Mutagénesis , Recombinasas , Recombinación Genética , Intercambio de Cromátides HermanasRESUMEN
The dif locus is a RecA-independent recombination site, located in the terminus region of the chromosome of Escherichia coli. This site functions to reduce circular dimer chromosomes to monomers before cell division. Strains lacking this site exhibit the Dif phenotype, in which a fraction of the cells form extended filaments with abnormal nucleoids, and the SOS system is induced. We have used a transposon (Tndif), as well as linear transformation, to position dif in 19 locations around the chromosome. All of the suppressing insertions that we obtained were within 10 kb of the normal site, even in strains in which the normal symmetry, between the origin of replication and dif had been altered by 200 kb. We also observed that the nonsuppressing insertions in the terminus region became suppressing if a deletion occurred that extended from the ectopic site up to or past the normal location of dif. We propose that dif is normally located at the center of converging polarities in the terminus region and that deletions that restore suppression do so by placing ectopic sites once again at the center of this polarity. Similar results and conclusions are described in this issue.
Asunto(s)
Cromosomas Bacterianos , ADN Nucleotidiltransferasas , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Recombinación Genética/genética , Replicación del ADN , Modelos Genéticos , Mutagénesis Insercional , Plásmidos/genética , Eliminación de Secuencia , Supresión Genética , Transformación Bacteriana , TransposasasRESUMEN
The dif locus (deletion-induced filamentation) of Escherichia coli is a resolvase site, located in the terminus region of the chromosome, that reduces chromosome multimers to monomers. In strains in which this site has been deleted, a fraction of the cells is filamentous, has abnormal nucleoid structure, and exhibits elevated levels of the SOS repair system. We have demonstrated that a 33-bp sequence, which is sufficient for RecA-independent recombination and which shows similarity to the cer site of pColE1, suppresses the Dif phenotype when inserted in the terminus region. Flanking sequences were not required, since suppression occurred in strains in which dif as well as 12 kb or 173 kb of DNA had been deleted. However, location was important, and insertions at a site 118 kb away from the normal site did not suppress the Dif phenotype. These sites were otherwise still functional, and they exhibited wild-type levels of RecA-independent recombination with dif-containing plasmids and recombined with other chromosomal dif sites to cause deletions and inversions. It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region.
Asunto(s)
ADN Bacteriano/genética , Escherichia coli/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , División Celular , Mapeo Cromosómico , Cromosomas Bacterianos/metabolismo , Replicación del ADN , Datos de Secuencia Molecular , Fenotipo , Recombinación Genética , Mapeo RestrictivoRESUMEN
'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.
Asunto(s)
Cromosomas Bacterianos/fisiología , Replicación del ADN , Escherichia coli/fisiología , Metiltransferasas/deficiencia , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica) , Proteínas Bacterianas/metabolismo , División Celular , ADN Bacteriano/análisis , Proteínas de Unión al ADN/metabolismo , Escherichia coli/citología , Proteínas de Escherichia coli , Modelos Genéticos , Mutación , Periodicidad , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
The tus gene encodes a DNA-binding protein (Tus) that inhibits replication forks at specific block-sites within the terminus region of the Escherichia coli chromosome. One of these block-sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregulates expression at this weak promoter. We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos/fisiología , Transcripción Genética/fisiología , Secuencia de Bases , Homeostasis/fisiología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Alquilación , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cromosomas Bacterianos , Dermatoglifia del ADN , Replicación del ADN , ADN Bacteriano/biosíntesis , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Semivida , Hidróxidos , Radical Hidroxilo , Cinética , Datos de Secuencia Molecular , PlásmidosRESUMEN
dif (deletion induced filamentation) is a newly identified locus that lies within the terminus region of the Escherichia coli chromosome. The Dif phenotype was characterized by a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system. Interactions between dif-carrying plasmids as well as between such plasmids and the bacterial chromosome demonstrated that dif is a cis-acting, recA-independent recombination site. Filamentation continued in dif mutants in which SOS-associated division inhibitors were inoperative, which showed that induction of these inhibitors was not the primary cause of filamentation. Filamentation was not observed in dif recA or dif recBC mutants, which were unable to carry out homologous recombination. The dif site shows homology with the cer site of plasmid ColE1, which resolves plasmid multimers to monomers. It is proposed that dif functions to resolve dimeric chromosomes produced by sister chromatid exchange, and that the Dif phenotype is due to the inability of these mutants to resolve multimers prior to cell division.
Asunto(s)
Deleción Cromosómica , Cromosomas Bacterianos , Escherichia coli/genética , Recombinación Genética , Regiones Terminadoras Genéticas , Secuencia de Bases , Mapeo Cromosómico , Elementos Transponibles de ADN , ADN Bacteriano/química , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Kanamicina/farmacología , Datos de Secuencia Molecular , FenotipoRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Replicación del ADN/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Datos de Secuencia Molecular , Salmonella typhimurium/genética , Homología de Secuencia de Ácido NucleicoAsunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Replicación del ADN , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Reguladores , Regiones Terminadoras Genéticas , Secuencia de Bases , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Inhibition sites T1 and T2 from the Escherichia coli terminus functioned with the same characteristics in ColE1-derived plasmids and in the chromosome. These characteristics included polarity and dependence on tus, a trans-acting factor required for inhibition. Inhibition in the terminus region of the R6K plasmid was also tus dependent.
Asunto(s)
Cromosomas Bacterianos , Replicación del ADN , Escherichia coli/genética , Genes Reguladores , Plásmidos , Regiones Terminadoras Genéticas , Mapeo Cromosómico , Mapeo RestrictivoRESUMEN
The components for termination of DNA replication in Escherichia coli include the terminator signals T1 and T2 and the trans-acting gene tus. We have shown previously that tus maps in a 4-kilobase region of the chromosomal terminus near T2. Through the use of deletion and insertion mutants, the location of the tus gene has now been precisely identified. We sequenced 2416 nucleotides in this region and identified a 927-base-pair open reading frame which encodes Tus. Insertion of a kanamycin-resistance gene in this open reading frame abolished tus activity. We also demonstrated that crude extracts of tus+ cells contain a protein which binds to the T2 terminator sequence.
Asunto(s)
Proteínas Bacterianas/genética , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Genes Reguladores , Genes , Regiones Terminadoras Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Prueba de Complementación Genética , Genotipo , Datos de Secuencia Molecular , Plásmidos , Mapeo RestrictivoRESUMEN
The terminus region of the E. coli chromosome contains two loci, T1 and T2, that inhibit the progress of replication forks and require the trans-acting factor tus. We have identified a 23 bp terminator signal at T1 and T2 that is within 100 bp of the sites of replication arrest. When an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly. We also found this terminator sequence in the terminus region of the plasmid R6K and in the origin region of RepFIIA class plasmids. In addition, we found striking similarities between the E. coli terminator signal and the terminator sequence of B. subtilis.
Asunto(s)
Replicación del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Secuencia de Bases , Southern Blotting , Cromosomas Bacterianos , ADN Bacteriano/biosíntesis , Endonucleasas , Datos de Secuencia Molecular , Plásmidos , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.
Asunto(s)
ADN Bacteriano/genética , Genes Bacterianos , Fijación del Nitrógeno/genética , Plásmidos , Rhizobium/genética , Simbiosis , Clonación Molecular , Fabaceae , Mutación , Fenotipo , Plantas MedicinalesRESUMEN
We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome. The assay was based on the properties of restriction fragments that contain stalled replication forks. Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouché (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982). Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called T1, which we located at kilobase 90. In each case we found only a single, precise site of arrest. Inhibition at T1 was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for T1 to function. We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells. In all previous studies on the terminus of E. coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition.