RESUMEN
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation¼ - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated¼ PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
Asunto(s)
Sistemas CRISPR-Cas , ADN Intergénico , Escherichia coli/genéticaRESUMEN
We found that mutations that increased the transcription of the mgtCBR (Mg2+ transport-related) operon conferred increased thermotolerance on this organism. The 5' leader of the mgtCBR mRNA contains two short open reading frames (ORFs), mgtM and mgtP, whose translation regulates the expression of the mgtCBR operon by a mechanism that is similar to attenuation in amino acid biosynthetic operons. We obtained two types of mutations that resulted in elevated transcription of the operon: defects in the mgtM ribosome-binding site, impairing the translation of this ORF and deletions encompassing the stop codon of mgtM that extend the translation of this ORF across a downstream Rho termination site. These mgtM mutations give further insights into the mechanism of the transcriptional control of the mgtCBR operon that we discuss in this work. We show that the increased thermotolerance requires elevated expression of the mgtC gene, but functional mgtB and mgtR, which respectively encode an Mg2+ transporter and a regulatory protein, are dispensable for this response. MgtC has been shown to have complex functions, including a requirement for virulence, flagella-independent motility and synthesis of cellulose and we now found that it has a role in the regulation of thermotolerance.
Asunto(s)
Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Transporte de Catión/metabolismo , Salmonella typhimurium/genética , Eliminación de Secuencia , Termotolerancia/genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Respuesta al Choque Térmico/genética , Sistemas de Lectura Abierta/genética , Operón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serogrupo , Virulencia/genéticaRESUMEN
CRISPR-Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR-Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR-Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR-Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR-Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release.
Asunto(s)
Bacteriólisis , Bacteriófagos/fisiología , Sistemas CRISPR-Cas , Escherichia coli/fisiología , Escherichia coli/virología , Bacteriófago lambda/genética , Marcación de Gen , Variación Genética , Genoma Viral , Fagos T/genéticaRESUMEN
During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , ADN/química , ADN/metabolismo , ADN Helicasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos/genéticaRESUMEN
In Salmonella enterica serovar Typhimurium, Mg2+ limitation induces transcription of the mgtA Mg2+ transport gene, but the mechanism involved is unclear. The 5' leader of the mgtA mRNA contains a 17-codon, proline-rich ORF, mgtL, whose translation regulates the transcription of mgtA [Park S-Y et al. (2010) Cell 142:737-748]. Rapid translation of mgtL promotes formation of a secondary structure in the mgtA mRNA that permits termination of transcription by the Rho protein upstream of mgtA, whereas slow or incomplete translation of mgtL generates a different structure that blocks termination. We identified the following mutations that conferred high-level transcription of mgtA at high [Mg2+]: (i) a base-pair change that introduced an additional proline codon into mgtL, generating three consecutive proline codons; (ii) lesions in rpmA and rpmE, which encode ribosomal proteins L27 and L31, respectively; (iii) deletion of efp, which encodes elongation factor EF-P that assists the translation of proline codons; and (iv) a heat-sensitive mutation in trmD, whose product catalyzes the m1G37 methylation of tRNAPro Furthermore, substitution of three of the four proline codons in mgtL rendered mgtA uninducible. We hypothesize that the proline codons present an impediment to the translation of mgtL, which can be alleviated by high [Mg2+] exerted on component(s) of the translation machinery, such as EF-P, TrmD, or a ribosomal factor. Inadequate [Mg2+] precludes this alleviation, making mgtL translation inefficient and thereby permitting mgtA transcription. These findings are a significant step toward defining the target of Mg2+ in the regulation of mgtA transcription.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Magnesio/química , Proteínas de Transporte de Membrana/metabolismo , Péptidos/química , Prolina/química , Salmonella typhimurium/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Codón , Proteínas de Escherichia coli/química , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Mutación , Factores de Elongación de Péptidos/química , Péptidos/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Biosíntesis de Proteínas , ARN de Transferencia/química , Ribosomas/química , Ribosomas/metabolismo , Transcripción Genética/efectos de los fármacos , ARNt Metiltransferasas/químicaRESUMEN
Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers-short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient "primed" adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector-crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference-that is, target destruction without acquisition of additional spacers-is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector-crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.
Asunto(s)
Sistemas CRISPR-Cas , ADN Intergénico , Escherichia coli/fisiología , Adaptación Biológica , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.
Asunto(s)
Escherichia coli/genética , Adaptación Fisiológica , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Interferencia de ARN , ARN Bacteriano/fisiologíaRESUMEN
CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.
Asunto(s)
Proteínas Asociadas a CRISPR/fisiología , Sistemas CRISPR-Cas , Endorribonucleasas/fisiología , Escherichia coli/genética , Bacteriófagos/genética , Proteínas Asociadas a CRISPR/metabolismo , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Plásmidos/genética , ARN/metabolismo , Terminación de la Transcripción GenéticaRESUMEN
CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Adaptación Fisiológica , Bacteriófagos/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Plásmidos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/virología , ARN Bacteriano/metabolismo , Proteínas Virales/metabolismoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5' handle (8 nt) and a 3' handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5' handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Asociadas a CRISPR/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Unión Proteica , ARN Guía de Kinetoplastida/químicaRESUMEN
During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that in Escherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/genética , Evolución Molecular , Secuencia de Bases , Secuencia Conservada , ADN Bacteriano/genética , ADN IntergénicoRESUMEN
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.
Asunto(s)
Inmunidad Adaptativa/genética , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Escherichia coli/genética , Emparejamiento Base/genética , ADN/genética , Motivos de Nucleótidos/genética , ARN/genéticaRESUMEN
Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.
Asunto(s)
Acetiltransferasas/metabolismo , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/toxicidad , Ácido Aspártico/análogos & derivados , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Iniciación de la Cadena Peptídica Traduccional , Acetiltransferasas/genética , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Ácido Aspártico/toxicidad , Bacteriocinas/química , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacosRESUMEN
Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
Asunto(s)
Secuencia de Bases , ARN/genética , ARN/metabolismo , ADN Viral/genética , ADN Viral/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Secuencias Invertidas Repetidas , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Virus/genética , Virus/inmunologíaRESUMEN
Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Péptidos/química , Transportadoras de Casetes de Unión a ATP/genética , Antibacterianos/química , Bacteriocinas/química , Transporte Biológico , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Estructura Molecular , Péptidos/genética , Péptidos/metabolismoRESUMEN
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ-phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently 'silent'E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
Asunto(s)
ADN Bacteriano/genética , Escherichia coli/genética , Secuencias Invertidas Repetidas , Transcripción Genética , Regiones no Traducidas 5' , Bacteriófago lambda/fisiología , Secuencia de Bases , ADN Intergénico/genética , Escherichia coli/inmunología , Escherichia coli/virología , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Procesamiento Postranscripcional del ARNRESUMEN
Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last 25 years. Genome-wide screening using the 'Keio collection' to test sensitivity to colicins (col) A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the lipopolysaccharide inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of lipopolysaccharide and enterobacterial common antigen. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Peptidoglycan-associated lipoprotein, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene product has the necessary proteolysis function.
Asunto(s)
Colicinas/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Colicinas/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Biblioteca de Genes , Genes Bacterianos , Prueba de Complementación GenéticaRESUMEN
Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ADN Bacteriano/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Microorganismos Modificados Genéticamente , TransgenesRESUMEN
The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.
Asunto(s)
Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptido Hidrolasas/metabolismo , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Aspartato-ARNt Ligasa/antagonistas & inhibidores , Aspartato-ARNt Ligasa/metabolismo , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometría de Masas , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Estructura Molecular , Mutación , Péptido Hidrolasas/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).