The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

In the process of CRISPR adaptation, short pieces of DNA ("spacers") are acquired from foreign elements and integrated into the CRISPR array. It so far 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 dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA 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 from both high copy plasmids and phages.

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibioticresistant pathogens with sensitive ones.CRISPR-Cas | positive selection | lysogenization | ex vivo treatment

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5′-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5′-AA, positioned at the 3′ end of the spacer, that enhances efficiency of the spacer's acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5′-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.defense mechanism | phage-host interaction | acquisition step C lustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) comprise an important prokaryotic defense system against horizontally transferred DNA (1-3) and RNA (4). This system shows remarkable analogies to the mammalian immune system (5, 6) and to eukaryotic RNA-interference mechanisms (7, 8). Three major types and 10 subtypes of CRISPR/Cas systems (9) have been found across ∼90% of archaeal genomes and ∼50% of bacterial genomes. All types consist of a CRISPR array-short repeated sequences called "repeats" flanking short sequences called "spacers." The array is usually preceded by a leader, AT-rich DNA sequence that drives CRISPR array expression and is important for acquiring new spacers into the array (10, 11). A cluster of CRISPR-associated (cas) genes encoding proteins that process the transcript, interfere with foreign nucleic acids, and acquire new spacers usually lies adjacent to the CRISPR array (12)(13)(14). RNA transcribed from the CRISPR array (crRNA) is processed by Cas proteins into RNA-based spacers flanked by partial repeats. These crRNAs specifically direct Cas interfering proteins to target nucleic acids matching the spacers. The spacers are acquired from these targeted sequences, termed "protospacers." Spacer acquisition into the CRISPR array consequently results in guiding the system to cleave DNA molecules harboring the corresponding protospacers. This feature renders the system competent in adaptively and specifically targeting invaders.Spacer acquisition into a CRISPR array...

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNAmediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.defense mechanism | phage-host interactions | positive selection T he clustered regularly interspaced short palindromic repeats (CRISPR) system is a significant defense mechanism of prokaryotes against viruses and horizontally transferred DNA (1-3) and RNA (4). It is found in ∼90% of archaeal genomes and ∼40% of bacterial genomes, and consists of a CRISPR array, usually preceded by a leader DNA sequence, located near a cluster of CRISPR-associated (cas) genes (5-7). RNA transcribed from the CRISPR array (crRNA) is processed by Cas proteins and directs interfering proteins to target DNA/RNA matching the sequences between the repeats. These sequences, called spacers, often originate from plasmids and phages; thus, the system adaptively targets these invaders. High variability is found among bacterial species in the number, sequence, and length of the CRISPR arrays; the sequence and length of the leader DNA; and the number and sequence of the associated proteins. The CRISPR arrays are composed of 2 to ∼250

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.host takeover | bacterial division | bacteriophage biology T he abundance of bacteriophages and their importance to microbial evolution, and consequently to major ecological issues, provide an incentive to study their biology. Bacteriophage T7 and its host, Escherichia coli, provide a model for systematically studying host-virus interactions. The genetics of both the phage and its host have been studied extensively, and the putative functions or tentative physiological roles of over half of the 56 genes of phage T7 and the 4,453 genes of E. coli have been identified (1-3). T7 is a virulent phage which, upon infection of its host E. coli, produces over 100 progeny phage per host in less than 25 min. It is an obligatory lytic phage, meaning that a successful phage growth cycle always results in lysis of the host. The genome of T7 is a 39,937-bp double-stranded DNA molecule (4). Despite extensive knowledge of phage T7, the mechanism by which it takes over the host molecular machinery remains obscure. Specific functions have been attributed to over

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.