Transient enhanced cell division by blocking DNA synthesis in Escherichia coli.

Duplication of the bacterial nucleoid is necessary for cell division hence specific arrest of DNA replication inhibits divisions culminating in filamentation, nucleoid dispersion and appearance of a-nucleated cells. It is demonstrated here that during the first 10 min however, Escherichia coli enhanced residual divisions: the proportion of constricted cells doubled (to 40%), nucleoids contracted and cells remodelled dimensions: length decreased and width increased. The preliminary data provides further support to the existence of temporal and spatial couplings between the nucleoid/replisome and the sacculus/divisome, and is consistent with the idea that bacillary bacteria modulate width during the division process exclusively.

Imaging of Transcription and Replication in the Bacterial Chromosome with Multicolor Three-Dimensional Superresolution Structured Illumination Microscopy.

Superresolution imaging technology has contributed to our understanding of the subnucleoid organization in E. coli cells. Multicolor superresolution images revealing "bacterial nucleolus-like structure or organization," "nucleolus-like compartmentalization of the transcription factories," and "spatial segregation of the transcription and replication machineries" have enhanced our understanding of the dynamic landscape of the bacterial chromatin. This chapter provides a brief introduction into multicolor three-dimensional superresolution structured illumination microscopy (3D-SIM) used to study the spatial organization of the transcription machinery and its spatial relationship with replisomes from a microbiological research perspective. In addition to a detailed protocol, practical considerations are discussed in relation to (1) sampling and treatment of cells containing fluorescent fusion proteins, (2) imaging the transcription and replication machineries at single-cell levels, (3) performing imaging experiments to capture the spatial organization of the transcription machinery and the nucleoid, and (4) image acquisition and analysis.

Rifampicin suppresses thymineless death by blocking the transcription-dependent step of chromosome initiation.

Thymineless death (TLD), a phenomenon in which thymine auxotrophy becomes lethal when cells are starved of thymine, can be prevented by the presence of rifampicin, an RNA polymerase inhibitor. Several lines of evidence link TLD to chromosome initiation events. This suggests that rifampicin-mediated TLD suppression could be due to the inhibition of RNA synthesis required for DNA chromosomal initiation at oriC, although other mechanisms cannot be discarded. In this work, we show that the addition of different rifampicin concentrations to thymine-starved cells modulates TLD and chromosomal initiation capacity (ChIC). Time-lapse experiments find increasing levels of ChIC during thymine starvation correlated with the accumulation of simple-Y, double-Y and bubble arc replication intermediates at the oriC region as visualized by two-dimensional DNA agarose gel electrophoresis. None of these structures were observed following rifampicin addition or under genetic-physiological conditions that suppress TLD, indicating that abortive chromosome replication initiations under thymine starvation are crucial for this lethality. Significantly, the introduction of mioC and gid mutations which alter transcription levels around oriC, reduces ChIC and alleviates TLD. These results show that the impairment of transcription-dependent initiation caused by rifampicin addition, is responsible for TLD suppression. Our findings here may provide new avenues for the development of improved antibacterial treatments and chemotherapies based on thymine starvation-induced cell death.

DNA replication initiation as a key element in thymineless death.

Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles (n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase.