RESUMEN
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.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Acetato CoA Ligasa/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporteros , Genómica/métodos , Regiones Promotoras GenéticasRESUMEN
Gene expression is controlled by the joint effect of (i) the global physiological state of the cell, in particular the activity of the gene expression machinery, and (ii) DNA-binding transcription factors and other specific regulators. We present a model-based approach to distinguish between these two effects using time-resolved measurements of promoter activities. We demonstrate the strength of the approach by analyzing a circuit involved in the regulation of carbon metabolism in E. coli. Our results show that the transcriptional response of the network is controlled by the physiological state of the cell and the signaling metabolite cyclic AMP (cAMP). The absence of a strong regulatory effect of transcription factors suggests that they are not the main coordinators of gene expression changes during growth transitions, but rather that they complement the effect of global physiological control mechanisms. This change of perspective has important consequences for the interpretation of transcriptome data and the design of biological networks in biotechnology and synthetic biology.
Asunto(s)
Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Carbono/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factor Proteico para Inverción de Estimulación/genética , Factor Proteico para Inverción de Estimulación/metabolismo , Redes Reguladoras de Genes , Reproducibilidad de los ResultadosRESUMEN
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.