It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and identified multiple families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that an anti-Gabija protein, denoted Gad1, blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.
Metabolic engineering is often facilitated by cloning of genes encoding enzymes from various heterologous organisms into E. coli. Such engineering efforts are frequently hampered by foreign genes that are toxic to the E. coli host. We have developed PanDaTox (www.weizmann.ac.il/pandatox), a web-based resource that provides experimental toxicity information for more than 1.5 million genes from hundreds of different microbial genomes. The toxicity predictions, which were extensively experimentally verified, are based on serial cloning of genes into E. coli as part of the Sanger whole genome shotgun sequencing process. PanDaTox can accelerate metabolic engineering projects by allowing researchers to exclude toxic genes from the engineering plan and verify the clonability of selected genes before the actual metabolic engineering experiments are conducted. Metabolic Engineering and the Problem of Toxic GenesMetabolic engineering is a rapidly growing field where enzymatic pathways for the biosynthesis of desired molecules are genetically engineered into model microorganisms in order to harness bacterial productivity into industrial use. 1Metabolic engineering is often practiced in close connection with synthetic biology and systems biology, as significant theoretical modeling is conducted in order to model pathways to be engineered into a given organism. 2-5The bacterial species Escherichia coli is one of the most widely used PanDaToxA tool for accelerated metabolic engineering Gil Amitai and Rotem Sorek* Department of Molecular Genetics; Weizmann Institute of Science; Rehovot, Israel microorganisms in metabolic engineering and had been utilized for numerous bio-production applications. For example, massive production of precursors for the antimalarial drug artimisinin was facilitated by inserting genes from several microorganisms into E. coli; 6,7 polyketides, which are the precursors for many antibiotics, have been produced within E. coli by introducing a combination of genes from three different bacteria into this organism;8 various nutritional molecules such as acetate, pyruvate, succinate and an array of amino acids are also among the products produced within E. coli through metabolic engineering.9 Recently, metabolic engineering has taken center stage in the global efforts for the design and generation of biofuel-producing organisms, in attempts to generate biological alternatives to fossil fuels. 1,10Thousands of microbial genomes have been sequenced to date 5,11 and these genomes cumulatively harbor millions of enzymes that can be used as "building blocks" in the toolbox of the metabolic engineer. Following detailed planning of the pathway of new enzymes to engineer into E. coli, the metabolic engineer will usually search for these enzymes in the set of available genomes and will select genes encoding the desired enzymes from organisms in which these genes exist. The selected genes will then be cloned into E. coli with or without optimization for expression through codon and promoter alterations. 3,12One of th...
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