Gil Amitai

Directed evolution by in vitro compartmentalization

aqueous droplets per ml of emulsion. The majority of droplets contain no more than a single gene along with all of the molecular machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype

CRISPR adaptation biases explain preference for acquisition of foreign DNA.

CRISPR–Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA (‘spacers’) are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

CRISPR-Cas adaptation: insights into the mechanism of action.

Since the first demonstration that CRISPR–Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR–Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR–Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

Communication between viruses guides lysis–lysogeny decisions

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses decide between the lytic and the lysogenic cycles, that is, whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the SPbeta group use a small-molecule communication system to coordinate lysis–lysogeny decisions. During infection of its Bacillus host cell, the phage produces a six amino-acids-long communication peptide that is released into the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We term this communication system the ‘arbitrium’ system, and further show that it is encoded by three phage genes: aimP, which produces the peptide; aimR, the intracellular peptide receptor; and aimX, a negative regulator of lysogeny. The arbitrium system enables a descendant phage to ‘communicate’ with its predecessors, that is, to estimate the amount of recent previous infections and hence decide whether to employ the lytic or lysogenic cycle.

Distribution of split DnaE inteins in cyanobacteria.

Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein‐encoded protein region is removed by an autocatalytic protein‐splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analysing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two‐genes one‐protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnology.

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C‐terminal region of Hedgehog and Hedgehog‐like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein‐splicing of their host‐protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N‐terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein‐like (BIL) domains. Bacterial intein‐like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C‐terminal flank and could also protein‐splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.

A vast collection of microbial genes that are toxic to bacteria

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

Systematic discovery of antiphage defense systems in the microbial pangenome

Maps of defense arsenals in microbial genomes To survive the attack of foreign invaders such as viruses and plasmids, bacteria and archaea fight back with immune systems that are usually clustered in “defense islands” in their genomes. Doron et al. took advantage of this property to map microbial defense systems systematically (see the Perspective by Kim). Candidate immune systems were then experimentally validated for their activities. Like well-known defense arsenals such as restriction-modification and CRISPR systems, these additional immune systems now require mechanistic investigation and could potentially be engineered into useful molecular tools in the future. Science, this issue p. eaar4120; see also p. 993 Bioinformatics and experimental validation identify nine antiphage and one antiplasmid immune defense systems in microbes. INTRODUCTION Bacteria and archaea are frequently attacked by viruses (phages) and as a result have developed multiple, sophisticated lines of active defense that can collectively be referred to as the prokaryotic “immune system.” Although bacterial defense against phages has been studied for decades, it was suggested that many currently unknown defense systems reside in the genomes of nonmodel bacteria and archaea and await discovery. RATIONALE Antiphage defense systems are known to be frequently physically clustered in microbial genomes such that, for example, genes encoding restriction enzymes commonly reside in the vicinity of genes encoding other phage resistance systems. The observation that defense systems are clustered in genomic “defense islands” has led to the hypothesis that genes of unknown function residing within such defense islands may also participate in antiphage defense. In this study, we aimed to comprehensively identify and experimentally verify new defense systems based on their enrichment within defense islands in an attempt to systematically map the arsenal of defense tools that are at the disposal of microbes in their fight against phages. RESULTS We searched for gene cassettes of unknown function that are enriched near known defense systems in more than 45,000 available bacterial and archaeal genome sequences. Such gene cassettes were defined as candidate defense systems and were systematically engineered into model bacteria, which were then infected by an array of phages to test for antiphage activities. This yielded the discovery of nine new families of antiphage defense systems and one additional family of antiplasmid systems that are widespread in microbes and shown to strongly protect against foreign DNA invasion. The systems discovered include ones that seem to have adopted components of the bacterial flagella and chromosome maintenance complexes and use these components for defensive capacities. Our data also show that genes with Toll-interleukin receptor (TIR) domains are involved in bacterial defense against phages, providing evidence for a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria. CONCLUSIONS Our study expands the known arsenal of defense systems used by prokaryotes for protection against phages, exposing tens of thousands of instances of defense systems that were so far unknown. Some of these systems appear to employ completely new mechanisms of defense against phages. In the past, the discovery and mechanistic understanding of antiphage defense systems led to the development of important biotechnological tools, as exemplified by the use of restriction enzymes and CRISPR-Cas for biotechnological and biomedical applications. One may envision that some of the systems discovered in the current study, once their mechanism is deciphered, will also be adapted into useful molecular tools in the future. A pipeline for systematic discovery of defense systems. Microbial genomes (more than 45,000 in the current study) are mined for genetic systems that are physically enriched next to known defense systems such as restriction-modification and CRISPR-Cas. Candidate predicted systems are cloned into model bacteria, and these bacteria are then infected by an array of phages from various families to determine whether they provide defense. The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in “defense islands” in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.

Repeat Size Determination by Two Molecular Rulers in the Type I-E CRISPR Array

Summary Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications.

PanDaTox: a tool for accelerated metabolic engineering.

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 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.