Thymineless death, at the origin.

Thymineless death (TLD) in bacteria has been a focus of research for decades. Nevertheless, the advances in the last 5 years, with Escherichia coli as the model organism, have been outstanding. Independent research groups have presented compelling results that establish that the initiation of chromosome replication under thymine starvation is a key element in the scenario of TLD. Here we review the experimental results linking the initiation of replication to the lethality under thymine starvation and the proposed mechanisms by which TLD occurs. The concept of this relationship was 'in the air,' but approaches were not sufficiently developed to demonstrate the crucial role of DNA initiation in TLD. Genome-wide marker frequency analysis and Two Dimensional agarose gel electrophoresis have been critical methods employed to reveal that initiation events and the degradation of the oriC region occur during thymine starvation. The relationships between these events and TLD have established them to be the main underlying causes of the lethality under thymine starvation. Furthermore, we summarize additional important findings from the study of different mutant strains, which support the idea that the initiation of chromosomal replication and TLD are connected.

The dynamic nature and territory of transcriptional machinery in the bacterial chromosome.

Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.