RESUMO
Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. However, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/metabolismo , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Modelos Genéticos , Simulação de Dinâmica Molecular , Origem de Replicação/genéticaRESUMO
RATIONALE: Polyethylene glycols (PEGs) are soluble molecules utilized in a wide range of applications. Mass spectrometry and fragmentation patterns of positively charged PEG oligomers are well-known, but decomposition mechanisms of the deprotonated ions have not been studied. METHODS: Deprotonated PEGs were generated by electrospray ionization of PEG in water/acetonitrile. Collision-induced dissociation (CID) experiments were carried out in a tandem mass spectrometer. The anions were studied using a tandem mass spectrometer to carry out CID experiments. A series of small PEG oligomers, with 1 to 8 monomer units, were studied in order to monitor size-dependent effects on fragmentation reactions. RESULTS: Because deprotonated PEG ions have a unique charge site, their dissociation pathways can easily be monitored. The ions fragment by loss of C2H4O monomer units, with an alternating intensity pattern that suggests the loss of an even number of monomer units is favored. Smaller oligomers and oligomer fragments also yielded fragments corresponding to H2 elimination and H2O loss. H2 elimination occurs by the generation of a hydride ion which deprotonates an alcohol upon leaving, while dehydration appears to be a charge-remote process. CONCLUSIONS: The fragmentation of deprotonated PEG is dominated by intramolecular S(N)2 reactions involving the terminal oxide anion.
RESUMO
Early investigations of protonated polyethylene glycol fragmentation suggested the dissociation mechanism includes both direct and sequential processes. Experiments designed to study the proposed mechanisms of sequential dissociation are absent from the literature. In order to obtain additional experimental details about the fragmentation reactions, the dissociation of protonated polyethylene glycol was studied by energy-dependent collision-induced dissociation (CID). Key fragment ions were separated by mass differences corresponding to the loss of single monomer units. Several fragment ions were also generated by in-source fragmentation and studied by CID. These experiments indicate the primary ions undergo sequential dissociation by the loss of either one or two monomer units. The results suggest that at least two different mechanisms must be considered to explain the sequential dissociation of protonated polyethylene glycols. The reaction involving the elimination of two subunits suggests the loss of a six-membered 1,4-dioxane product, while the elimination of a single subunit involves the loss of acetaldehyde by a 1,2-hydride shift rearrangement.
