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

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Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed novel TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

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

Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterialgrowth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms. in some bacteria, the resistance mechanisms against most conventional antibiotics have been identified (1, 2). This increasing threat is spurring the identification of novel antimicrobials against novel molecular targets in the pathogens (e.g., refs. 3-6). There are currently only a few host molecules targeted by antibiotics. These targets (and examples of the antibiotics against them) are host RNA polymerase (rifampicin), topoisomerase (quinolones), cell wall (penicillin), membranes (polymyxin), ribosome (tetracyclines, aminoglycosides, macrolids), and synthesis of nucleic-acid precursors (sulfonamides, trimethoprim). Increasing the arsenal of bacterial targets and antimicrobial drugs against them is valuable, and novel strategies to increase this repertoire are therefore of great importance.One strategy for the identification of novel antibacterial targets is to determine how bacteriophages shut down their host's biosynthetic pathways and enslave its machinery during infection. Phages have coevolved with bacteria for over 3 billion years and have thus developed molecules to specifically and optimally inhibit or divert key metabolic functions. Examples of bacterial targets inhibited by phage-derived products include the δ subunit of the DNA polymerase III clamp loader, inhibited by gene product (Gp) 8 of the coliphage N4 (7); the Staphylococcus aureus putative helicase loader, DnaI, inhibited by ORF104 of bacteriophage 77 (5); a key enzyme of folate metabolism, FolD, inhibited by Gp55.1 of the coliphage T4 (8); and the essential cell-division protein, filamenting temperature-sen...

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The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.