RESUMEN
Of all known transposable elements, phage Mu exhibits the highest transposition efficiency and the lowest target specificity. In vitro, MuB protein is responsible for target choice. In this work, we provide a comprehensive assessment of the genome-wide distribution of MuB and its relationship to Mu target selection using high-resolution Escherichia coli tiling DNA arrays. We have also assessed how MuB binding and Mu transposition are influenced by chromosome-organizing elements such as AT-rich DNA signatures, or the binding of the nucleoid-associated protein Fis, or processes such as transcription. The results confirm and extend previous biochemical and lower resolution in vivo data. Despite the generally random nature of Mu transposition and MuB binding, there were hot and cold insertion sites and MuB binding sites in the genome, and differences between the hottest and coldest sites were large. The new data also suggest that MuB distribution and subsequent Mu integration is responsive to DNA sequences that contribute to the structural organization of the chromosome.
Asunto(s)
Bacteriófago mu/genética , Cromosomas/genética , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Bacteriófago mu/metabolismo , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Cromosomas/química , ADN/química , ADN/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/deficiencia , Factor Proteico para Inverción de Estimulación/genética , Dispositivos Laboratorio en un Chip , Mutagénesis Insercional , Técnicas de Amplificación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos , Unión Proteica/genética , Eliminación de SecuenciaRESUMEN
The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Factor Proteico para Inverción de Estimulación/metabolismo , Regulación de la Expresión Génica , Porinas/biosíntesis , Secuencia de Bases , Electroforesis en Gel Bidimensional , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/deficiencia , Factor Proteico para Inverción de Estimulación/genética , Eliminación de Gen , Datos de Secuencia Molecular , Mutación , Proteoma/análisisRESUMEN
How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter-lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [R(f)], and the ratio of the Michaelis-Menten parameters, V(max)/K(m) (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD(460)) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD(460) and corresponds to an intracellular concentration of about 50 microM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.