RESUMEN
Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.
Asunto(s)
Enzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Procesamiento Proteico-Postraduccional , Sitios de Unión/genética , Enzimas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Espectrometría de Masas/métodos , Metabolómica/métodos , Mutación , Fosforilación , Proteoma/metabolismo , Proteómica/métodos , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.
Asunto(s)
Bacteriófago lambda/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Exodesoxirribonucleasa V/metabolismo , Exonucleasas/metabolismo , Mycobacterium tuberculosis/genética , Secuencia de Bases , Sistema Libre de Células , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Exodesoxirribonucleasa V/antagonistas & inhibidores , Exonucleasas/antagonistas & inhibidores , Genes Bacterianos , Plásmidos/genética , Proteínas Recombinantes/metabolismo , Biología Sintética/métodos , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
While horizontal gene transfer is prevalent across the biosphere, the regulatory features that enable expression and functionalization of foreign DNA remain poorly understood. Here, we combine high-throughput promoter activity measurements and large-scale genomic analysis of regulatory regions to investigate the cross-compatibility of regulatory elements (REs) in bacteria. Functional characterization of thousands of natural REs in three distinct bacterial species revealed distinct expression patterns according to RE and recipient phylogeny. Host capacity to activate foreign promoters was proportional to their genomic GC content, while many low GC regulatory elements were both broadly active and had more transcription start sites across hosts. The difference in expression capabilities could be explained by the influence of the host GC content on the stringency of the AT-rich canonical σ70 motif necessary for transcription initiation. We further confirm the generalizability of this model and find widespread GC content adaptation of the σ70 motif in a set of 1,545 genomes from all major bacterial phyla. Our analysis identifies a key mechanism by which the strength of the AT-rich σ70 motif relative to a host's genomic GC content governs the capacity for expression of acquired DNA. These findings shed light on regulatory adaptation in the context of evolving genomic composition.
Asunto(s)
Bacterias , Transferencia de Gen Horizontal , Bacterias/genética , Composición de Base , ADN , Genoma Bacteriano/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.
Asunto(s)
Actinobacteria/genética , Firmicutes/genética , Ensayos Analíticos de Alto Rendimiento , Proteobacteria/genética , Fracciones Subcelulares/metabolismo , Transcripción Genética , Actinobacteria/química , Actinobacteria/metabolismo , Firmicutes/química , Firmicutes/metabolismo , Biblioteca de Genes , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteobacteria/química , Proteobacteria/metabolismo , Fracciones Subcelulares/química , Biología Sintética/métodosRESUMEN
Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here we report the large-scale metagenomic mining of thousands of natural 5' regulatory sequences from diverse bacteria, and their multiplexed gene expression characterization in industrially relevant microbes. We identified sequences with broad and host-specific expression properties that are robust in various growth conditions. We also observed substantial differences between species in terms of their capacity to utilize exogenous regulatory sequences. Finally, we demonstrate programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes. Together, these findings provide a rich resource of characterized natural regulatory sequences and a framework that can be used to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties, and also suggest the prospect of ultimately engineering complex behaviors at the community level.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Metagenómica/métodos , Elementos Reguladores de la Transcripción/fisiología , Minería de Datos , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Ingeniería Metabólica , Redes y Vías Metabólicas , Especificidad de la Especie , Biología Sintética/métodosRESUMEN
Advances in synthetic biology to build microbes with defined and controllable properties are enabling new approaches to design and program multispecies communities. This emerging field of synthetic ecology will be important for many areas of biotechnology, bioenergy and bioremediation. This endeavor draws upon knowledge from synthetic biology, systems biology, microbial ecology and evolution. Fully realizing the potential of this discipline requires the development of new strategies to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities. Here, we review recent experimental, analytical and computational advances to study and build multi-species microbial communities with defined functions and behavior for various applications. We also highlight outstanding challenges and future directions to advance this field.
Asunto(s)
Biodegradación Ambiental , Consorcios Microbianos/genética , Interacciones Microbianas/fisiología , Organismos Modificados Genéticamente/metabolismo , Biología Sintética/métodos , Biología de Sistemas/métodos , Biotecnología , Ecosistema , Ingeniería Genética , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/crecimiento & desarrolloRESUMEN
Understanding the mechanisms that generate variation is a common pursuit unifying the life sciences. Bacteria represent an especially striking puzzle, because closely related strains possess radically different metabolic and ecological capabilities. Differences in protein repertoire arising from gene transfer are currently considered the primary mechanism underlying phenotypic plasticity in bacteria. Although bacterial coding plasticity has been extensively studied in previous decades, little is known about the role that regulatory plasticity plays in bacterial evolution. Here, we show that bacterial genes can rapidly shift between multiple regulatory modes by acquiring functionally divergent nonhomologous promoter regions. Through analysis of 270,000 regulatory regions across 247 genomes, we demonstrate that regulatory "switching" to nonhomologous alternatives is ubiquitous, occurring across the bacterial domain. Using comparative transcriptomics, we show that at least 16% of the expression divergence between Escherichia coli strains can be explained by this regulatory switching. Further, using an oligonucleotide regulatory library, we establish that switching affects bacterial promoter architecture. We provide evidence that regulatory switching can occur through horizontal regulatory transfer, which allows regulatory regions to move across strains, and even genera, independently from the genes they regulate. Finally, by experimentally characterizing the fitness effect of a regulatory transfer on a pathogenic E. coli strain, we demonstrate that regulatory switching elicits important phenotypic consequences. Taken together, our findings expose previously unappreciated regulatory plasticity in bacteria and provide a gateway for understanding bacterial phenotypic variation and adaptation.