Density of σ70 promoter-like sites in the intergenic regions dictates the redistribution of RNA polymerase during osmotic stress in Escherichia coli.

RNA polymerase (RNAP), the transcription machinery, shows dynamic binding across the genomic DNA under different growth conditions. The genomic features that selectively redistribute the limited RNAP molecules to dictate genome-wide transcription in response to environmental cues remain largely unknown. We chose the bacterial osmotic stress response model to determine genomic features that direct genome-wide redistribution of RNAP during the stress. Genomic mapping of RNAP and transcriptome profiles corresponding to the different temporal states after salt shock were determined. We found rapid redistribution of RNAP across the genome, primarily at σ70 promoters. Three subsets of genes exhibiting differential salt sensitivities were identified. Sequence analysis using an information-theory based σ70 model indicates that the intergenic regions of salt-responsive genes are enriched with a higher density of σ70 promoter-like sites than those of salt-sensitive genes. In addition, the density of promoter-like sites has a positive linear correlation with RNAP binding at different salt concentrations. The RNAP binding contributed by the non-initiating promoter-like sites is important for gene transcription at high salt concentration. Our study demonstrates that hyperdensity of σ70 promoter-like sites in the intergenic regions of salt-responsive genes drives the RNAP redistribution for reprograming the transcriptome to counter osmotic stress.

A synthetic growth switch based on controlled expression of RNA polymerase.

The ability to control growth is essential for fundamental studies of bacterial physiology and biotechnological applications. We have engineered an Escherichia coli strain in which the transcription of a key component of the gene expression machinery, RNA polymerase, is under the control of an inducible promoter. By changing the inducer concentration in the medium, we can adjust the RNA polymerase concentration and thereby switch bacterial growth between zero and the maximal growth rate supported by the medium. We show that our synthetic growth switch functions in a medium-independent and reversible way, and we provide evidence that the switching phenotype arises from the ultrasensitive response of the growth rate to the concentration of RNA polymerase. We present an application of the growth switch in which both the wild-type E. coli strain and our modified strain are endowed with the capacity to produce glycerol when growing on glucose. Cells in which growth has been switched off continue to be metabolically active and harness the energy gain to produce glycerol at a twofold higher yield than in cells with natural control of RNA polymerase expression. Remarkably, without any further optimization, the improved yield is close to the theoretical maximum computed from a flux balance model of E. coli metabolism. The proposed synthetic growth switch is a promising tool for gaining a better understanding of bacterial physiology and for applications in synthetic biology and biotechnology.

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.

A genome-wide screen for identifying all regulators of a target gene.

We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP-cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.

WellReader: a MATLAB program for the analysis of fluorescence and luminescence reporter gene data.

MOTIVATION: Fluorescent and luminescent reporter gene systems in combination with automated microplate readers allow real-time monitoring of gene expression on the population level at high precision and sampling density. This generates large amounts of data for the analysis of which computer tools are missing to date. RESULTS: We have developed WellReader, a MATLAB program for the analysis of fluorescent and luminescent reporter gene data. WellReader allows the user to load the output files of microplate readers, remove outliers, correct for background effects and smooth and fit the data. Moreover, it computes biologically relevant quantities from the measured signals, notably promoter activities and protein concentrations, and compares the resulting expression profiles of different genes under different conditions. AVAILABILITY: WellReader is available under a LGPL licence at http://prabi1.inrialpes.fr/trac/wellreader.