RESUMO
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.
Assuntos
Bacteriófago T7/genética , DNA Bacteriano/genética , DNA Viral/genética , Escherichia coli/genética , Vetores Genéticos , Klebsiella pneumoniae/genética , Shigella sonnei/genética , Transdução Genética/métodos , Vírion , DNA Bacteriano/biossíntese , DNA Viral/biossíntese , Escherichia coli/metabolismo , Escherichia coli/virologia , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/virologia , Shigella sonnei/metabolismo , Shigella sonnei/virologiaRESUMO
Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.
Assuntos
Proteínas Associadas a CRISPR , Proteínas de Escherichia coli , Escherichia coli , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genéticaRESUMO
Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.
Assuntos
Bacteriófagos/genética , Bacteriófagos/fisiologia , DNA/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Pontos de Checagem do Ciclo Celular , Divisão Celular , Endonucleases , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Uracila/metabolismoRESUMO
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.
Assuntos
Bacteriófago T7/fisiologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/virologia , Plasmídeos , Proteínas Virais/genéticaRESUMO
Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.
Assuntos
Cromossomos de Mamíferos , Edição de Genes/métodos , Genoma , Processos de Determinação Sexual , Razão de Masculinidade , Animais , Cruzamento , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cruzamentos Genéticos , Feminino , Fertilização , Masculino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
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.
Assuntos
Adaptação Fisiológica , Bacteriófagos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Bacteriano/genética , DNA Viral/genética , Escherichia coli/genética , Plasmídeos/genética , Sistemas CRISPR-Cas/genética , Sequência Consenso/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA/genética , Exodesoxirribonuclease V/metabolismo , Modelos BiológicosRESUMO
We recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.
Assuntos
DNA Bacteriano/genética , Evasão da Resposta Imune/genética , Mutação/genética , Transdução Genética , Sequência de Bases , Epigênese Genética , Plasmídeos/genética , Reprodutibilidade dos TestesRESUMO
Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
Assuntos
Bacteriófago T7/metabolismo , Bacteriófago T7/patogenicidade , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Mutagênese , Dobramento de Proteína , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The extensive use of antibiotics over the last century has resulted in a significant artificial selection pressure for antibiotic-resistant pathogens to evolve. Various strategies to fight these pathogens have been introduced including new antibiotics, naturally-derived enzymes/peptides that specifically target pathogens and bacteriophages that lyse these pathogens. A new tool has recently been introduced in the fight against drug-resistant pathogens-the prokaryotic defense mechanism-clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system. The CRISPR-Cas system acts as a nuclease that can be guided to cleave any target DNA, allowing sophisticated, yet feasible, manipulations of pathogens. Here, we review pioneering studies that use the CRISPR-Cas system to specifically edit bacterial populations, eliminate their resistance genes and combine these two strategies in order to produce an artificial selection pressure for antibiotic-sensitive pathogens. We suggest that intelligent design of this system, along with efficient delivery tools into pathogens, may significantly reduce the threat of antibiotic-resistant pathogens.
Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/instrumentação , Edição de Genes/métodosRESUMO
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)-CRISPR-associated (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 antibiotic-resistant pathogens with sensitive ones.
Assuntos
Bactérias/virologia , Bacteriófagos/fisiologia , Farmacorresistência Bacteriana , Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Lisogenia , PlasmídeosRESUMO
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 bacterial-growth 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.
Assuntos
Bacteriófago T7/metabolismo , DNA Viral/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Genoma Viral/fisiologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , DNA Viral/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas Virais/genéticaRESUMO
Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.
Assuntos
Bacteriófagos/fisiologia , Interação Gene-Ambiente , Mutagênese , Fenótipo , Estresse Fisiológico , Bactérias/virologia , Seleção GenéticaAssuntos
Imunidade Adaptativa , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Viral/genética , DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/imunologia , Integrases/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genéticaRESUMO
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.
Assuntos
Adaptação Fisiológica/genética , DNA Bacteriano/genética , Escherichia coli/genética , Motivos de Nucleotídeos , Escherichia coli/fisiologiaRESUMO
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.
Assuntos
Adaptação Biológica/genética , Bacteriófago T7/genética , Proteínas do Citoesqueleto/antagonistas & inibidores , Escherichia coli/virologia , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Proteínas do Citoesqueleto/genética , Escherichia coli/citologia , Plasmídeos/genética , Proteínas Virais/genéticaRESUMO
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.
Assuntos
DNA Bacteriano/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Sequências Repetidas Invertidas , Adaptação Fisiológica , Escherichia coli/metabolismo , Motivos de NucleotídeosRESUMO
Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated 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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Choque Térmico HSP90/metabolismo , Temperatura Alta , Sequências Repetidas Invertidas/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Testes Genéticos , Proteínas de Choque Térmico HSP90/genética , Mutação/genética , Plasmídeos/genética , Prófagos/metabolismo , Reprodutibilidade dos TestesRESUMO
Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.
Assuntos
Bacteriófago T7 , Bacteriófagos , Bacteriófago T7/genética , Lipopolissacarídeos/metabolismo , Bacteriófagos/genética , Ligação Proteica , Proteínas de Transporte/metabolismo , Especificidade de HospedeiroRESUMO
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.