Although transcriptional regulation of gene expression during bacterial growth and stress response has been extensively studied, we have only recently begun to gain insights into how the transcription machinery is spatially organized in the nucleoid in response to growth conditions. Our study is the first that used fluorescent RNAP to image RNAP in E. coli. We found that the distribution of RNAP is dynamic and sensitive to environmental cues. We also discovered that, in fast-growing cells, RNAP concentrates as the predominant foci for active rRNA synthesis at clusters of rrn genes for ribosome RNA synthesis, resembling the eukaryotic nucleolus. Results from the microfluidic live-cell imaging system further validate the biological significance of RNAP foci and demonstrate the dynamic nature of the transcription machinery's organization. We also showed that active rRNA synthesis is the driving force for RNAP distribution in the cell and that active rRNA synthesis from multiple rrn operons is necessary for RNAP foci formation. Our work supports the idea that growth-rate regulation reflects the competition of RNAP distribution between rrn and other vast regions in the genome for limited RNAP in the cell. Co-imaging of RNAP and the nucleoid (DNA) in cells under different physiological conditions demonstrates that transcription by RNAP plays an important role in the nucleoid's organization. We performed the first E. coli genome conformation capture analysis, and the results revealed that the nucleoid is organized by both replication via SeqA-mediated interactions and transcription through gene clustering. Using super-resolution imaging systems, we showed that the transcription machinery is spatially organized and segregated from the replisome in fast-growing cells, which explains why the two important cellular functions remain in harmony in the cell. Our current and future research focuses on testing the following hypotheses: (i) RNAP foci are transcription factories or hubs for the expression of growth-promoting genes, which are spatially segregated from transcription silencing territories in the chromosome, and (ii) transcription factories and transcription-silencing territories are reorganized in response to environmental signals. As an example of studying stress responses, we have concentrated on osmotic stress response, which is a conserved process from bacteria to eukaryotes. An unsolved issue in the field is whether the RNAP-DNA interaction is sensitive to high salt in vivo. We co-imaged RNAP and DNA, in time-course experiments, in cells after high-salt shock and found that RNAP dissociates from the nucleoid during the initial high-salt shock when cytoplasmic K+ increases transiently, followed by RNAP reassociation during the later adaptation phase when K+ decreases. In parallel with the dissociation and reassociation of RNAP, there are significant changes in nucleoid structure. For current and future studies, we will (i) determine the mechanism(s) underlying the dynamic interaction between RNAP and promoter sequences during osmotic stress response in vivo; (ii) study the changes in transcriptome during the stress; and (iii) probe the spatial organization of transcription machinery for the osmotic stress-responsive genes. Together, these studies will provide an integrated view of transcriptional regulation of osmotic stress response in the whole E. coli system. We also extend our basic research to human pathogenic bacteria, mainly H. pylori, which is classified as a Group 1 carcinogen by the World Health Organization. We studied the role of SpoT in cell growth during serum starvation and found that SpoT is important in maintaining cell vitality. SpoT mediates the accumulation of polyphosphate (polyP) during serum starvation, at which time polyP binds to the principal sigma factor, sigma 80, to form highly stable complexes. The target site for the polyP binding is the unique arginine-rich N-terminal region (named the P region) of sigma 80. The putative P region is also found in the major sigma factors of other human pathogens, suggesting a new paradigm for pathogenesis regulation. We continue to study the interaction between sigma 80 and polyP, the enzyme(s) involved in the metabolism of polyP, and other SpoT-mediated stress responses. Our major effort in the future is to develop an in vitro transcription system, which is an important but challenging task because previous efforts on to do so have not succeeded. We also found that SpoT is important for H. pylori survival in macrophages; we will determine why, and analyze the changes in transcriptomes of both macrophages and H. pylori during infection. Moreover, we will test the hypothesis that genetic and epigenetic variations in H. pylori strains may influence associations with malignancy, in collaboration with Dr. Charles Rabkin, M.D., NCI Division of Cancer Epidemiology and Genetics (DCEG).
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