RESUMEN
A proposal is presented for classifying bacterial cell cycles into twelve discrete groups. This classification translates the three temporal parameters that define a cell cycle into numbers that facilitate an algorithmic approach to analyse the replication state of a single bacterium and of a bacterial population during steady-state of exponential growth. The classification and its implementation offer easy ways to obtain the rate of DNA synthesis and the amount of DNA per cell at any age in batch cultures.
Asunto(s)
Algoritmos , Ciclo Celular/fisiología , Escherichia coli/citología , Modelos Biológicos , Ciclo Celular/genética , División Celular/genética , División Celular/fisiología , Cromosomas Bacterianos/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN Bacteriano/clasificación , ADN Bacteriano/genética , Escherichia coli/genética , Cinética , Factores de TiempoRESUMEN
An algorithm is presented to determine the chromosome replication status, the rate of DNA synthesis per fork, and the amount of DNA in chromosome equivalents (G) per chromosome, per cell and per age throughout a bacterial cell cycle. This algorithm is the only attempt to study replication and the G value at any cell age since the general model of the bacterial cell cycle by Cooper and Helmstetter (1968, J. Mol. Biol. 31, 619-644). To help using it, two implementations are provided.
Asunto(s)
Ciclo Celular , Cromosomas Bacterianos , ADN Bacteriano/metabolismoRESUMEN
Ribonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that forkâ:âfocus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.
Asunto(s)
División Celular , Cromosomas/metabolismo , Replicación del ADN , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas Bacterianas/análisis , Citoplasma/química , ADN Polimerasa III/análisis , Proteínas de Unión al ADN/análisis , Escherichia coli/genética , Proteínas de Escherichia coli/análisisRESUMEN
BACKGROUND: There has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins. RESULTS: We tagged NrdB protein with 3xFLAG epitope and detected its subcellular location by immunofluorescence microscopy. We found that this protein is located in nucleoid-associated clusters, that the number of foci correlates with the number of replication forks at any cell age, and that after the replication process ends the number of cells containing NrdB foci decreases.We show that the number of NrdB foci is very similar to the number of SeqA, DnaB, and DnaX foci, both in the whole culture and in different cell cycle periods. In addition, interfoci distances between NrdB and three replication proteins are similar to the distances between two replication protein foci. CONCLUSIONS: NrdB is present in nucleoid-associated clusters during the replication period. These clusters disappear after replication ends. The number of these clusters is closely related to the number of replication forks and the number of three replication protein clusters in any cell cycle period. Therefore we conclude that NrdB protein, and most likely RNR protein, is closely linked to the replication proteins or replisome at the replication fork. These results clearly support the replication hyperstructure model.
Asunto(s)
Escherichia coli/enzimología , Ribonucleósido Difosfato Reductasa/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , AdnB Helicasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopía Fluorescente , Subunidades de Proteína/metabolismoRESUMEN
Bacterial cells contain many large, spatially extended assemblies of ions, molecules, and macromolecules, called hyperstructures, that are implicated in functions that range from DNA replication and cell division to chemotaxis and secretion. Interactions between these hyperstructures would create a level of organization intermediate between macromolecules and the cell itself. To explore this level, a taxonomy is needed. Here, we describe classification criteria based on the form of the hyperstructure and on the processes responsible for this form. These processes include those dependent on coupled transcription-translation, protein-protein affinities, chromosome site-binding by protein, and membrane structures. Various combinations of processes determine the formation, maturation, and demise of many hyperstructures that therefore follow a trajectory within the space of classification by form/process. Hence a taxonomy by trajectory may be desirable. Finally, we suggest that working toward a taxonomy based on speculative interactions between hyperstructures promises most insight into life at this level.
Asunto(s)
Bacterias/clasificación , Bacterias/citología , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Metabolismo Energético , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Bacteriano/genética , Desoxirribonucleótidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Replicación del ADN , ADN Bacteriano/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismoRESUMEN
The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Bacterias/citología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Redes y Vías MetabólicasRESUMEN
The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec(+) context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA(+) strain growing under limited (2-microg/ml) or under optimal (5-microg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.
Asunto(s)
Replicación del ADN , Escherichia coli/enzimología , Escherichia coli/fisiología , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/fisiología , División Celular/genética , Recuento de Colonia Microbiana , Roturas del ADN de Doble Cadena , Replicación del ADN/genética , ADN Bacteriano/metabolismo , Desoxirribonucleótidos/biosíntesis , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exodesoxirribonucleasa V/genética , Resolvasas de Unión Holliday/fisiología , Viabilidad Microbiana , MutaciónRESUMEN
NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA (ts) mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.
Asunto(s)
Segregación Cromosómica , Cromosomas Bacterianos/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/enzimología , Mutación/genética , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Temperatura , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Segregación Cromosómica/efectos de los fármacos , Segregación Cromosómica/efectos de la radiación , ADN Bacteriano/biosíntesis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/metabolismo , Hidroxiurea/farmacología , Indoles , Luz , Ribonucleósido Difosfato Reductasa/metabolismo , Dispersión de RadiaciónRESUMEN
An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR). Analysis of HIR in bacterial strains that had complete or partial oriC deletions and were suppressed by F integration showed that no sequence outside oriC is used for HIR. Analysis of a number of oriC mutants showed that deletion of the L-13-mer, which makes oriC inactive for cyclic replication, was the only mutation studied that inactivated HIR. The requirement for this sequence was strictly correlated with Benham's theoretical stress-induced DNA duplex destabilization. oriC mutations at DnaA, FIS, or IHF binding sites showed normal HIR activation, but DnaA was required for HIR. We suggest that strand opening for HIR initiation occurs due to heat-induced destabilization of the L-13-mer, and the stable oligomeric DnaA-single-stranded oriC complex might be required only to load the replicative helicase DnaB.
Asunto(s)
Proteínas Bacterianas/fisiología , Cromosomas Bacterianos/genética , Replicación del ADN , Proteínas de Unión al ADN/fisiología , Escherichia coli K12/fisiología , Regulación Bacteriana de la Expresión Génica , Complejo de Reconocimiento del Origen/fisiología , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Escherichia coli K12/genética , Calor , Complejo de Reconocimiento del Origen/genética , Mutación PuntualRESUMEN
Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.