RESUMEN
Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, we present a high-throughput infection method in a microplate reader, where the growth dynamics of the infected culture is measured using the optical density (OD). We find that the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers over nine orders of magnitude and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model allows us to infer the phage growth rate, which is a function of the phage-cell encounter rate, latent period, and burst size. Adding antibiotic selection provides the ability to measure the rate of host lysogenization. Using this method, we found that when E. coli growth slows down, the lytic growth rate of lambda phages decreases, and the propensity for lysogeny increases, demonstrating how host physiology influences the viral developmental program.
Asunto(s)
Escherichia coli , Lisogenia , Escherichia coli/virología , Escherichia coli/crecimiento & desarrollo , Bacteriófagos/fisiología , Bacteriófago lambda/fisiología , Bacteriófago lambda/genética , Modelos Biológicos , Dinámica Poblacional , Antibacterianos/farmacología , Modelos TeóricosRESUMEN
In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we developed a spatially resolved supercoiling model of transcription. The integrated model describes how RNAP activity feeds back with the local DNA supercoiling and how this mechanochemical feedback controls transcription, subject to topoisomerase activities and stochastic topological domain formation. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes, and this cooperation is achieved under moderate supercoiling diffusion and high topoisomerase unbinding rates. It predicts that a topological domain could serve as a transcription regulator, generating substantial transcriptional noise. It also shows the relative orientation of two closely arranged genes plays an important role in regulating their transcription. The model provides a quantitative platform for investigating how genome organization impacts transcription.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Transcripción Genética/genéticaRESUMEN
Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.