Section: New Results
Experimental mapping of gene regulatory networks in bacteria
The use of fluorescent and luminescent reporter genes allows real-time monitoring of gene expression, both at the level of individual cells and cell populations (Section 3.3 ). In order to fully exploit this technology, we need methods to rapidly construct reporter genes, both on plasmids and on the chromosome, mathematical models to infer biologically relevant quantities from the primary data, and computer tools to achieve this in an efficient and user-friendly manner. For instance, in a typical microplate experiment, 96 cultures are followed in parallel, over several hours, resulting in 10,000-100,000 measurements of absorbance and fluorescence and luminescence intensities. Over the past few years, we put into place an experimental platform and data analysis software, notably the WellReader program (Section 5.2 ), to allow biologists to make the most of the information contained in reporter gene expression data. Several improvements of the platform for measuring gene expression are the subject of ongoing work, including a novel method for efficiently cloning reporter gene constructions on the chromosome of E. coli.
These tools are actually used in a series of studies directed at the experimental mapping of gene regulatory networks in E. coli. One example, carried out in the framework of the PhD thesis of Guillaume Baptist, is the development of 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. Instead of a static picture of gene expression, this method allows dynamical monitoring in different environmental conditions. Mutants with interesting phenotypes can thus be selected based on multiple criteria, and the expression dynamics of the target gene can be extensively characterized. The method has been applied to the identification of the direct and indirect regulators of the gene acs in Escherichia coli. We confirmed known genetic regulators of the gene and identified new regulatory influences, many of which involve metabolic intermediates or metabolic sensing. An analysis of mutants involved in glycolysis and glucose transport demonstrates that the classical model of catabolite repression in E. coli needs to be amended. A paper describing the above work is currently under revision.
Other examples of on-going work are the analysis of the network involved in motility and sessility and the modulation of the RpoS regulon in E. coli by Omaya Dudin and Stephan Lacour, the validation of a model of the network of global regulators of transcription by Sara Berthoumieux and Hidde de Jong, and the analysis of the regulation of cAMP levels in the bacterial cell by Claire Villiers.