RESUMEN
In this communication, we report the adaptation of the CRISPR-Cas9 technology in Ustilago trichophora prototrophic wild-type isolate obtained from its natural host Echinochloa crus-galli. The established CRISPR vector and method enable a rapid and marker-free introduction of Cas9-induced non-homologous end-joining (NHEJ) dependent mutation at the targeted gene. Moreover, the method allows a specific modification of the chromosomal DNA sequence by Cas9-induced homologous recombination using short DNA repair templates. The results demonstrate the applicability of the CRISPR-Cas9 technology in U. trichophora for both gene knock-out by the NHEJ pathway and specific gene modification by templated genome editing, paving the way for rapid metabolic engineering of this Ustilago species for industrial applications.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica , Ustilago/genética , Secuencia de Bases , Cromosomas Fúngicos/genética , Marcadores Genéticos , Genoma Fúngico , Recombinación Homóloga/genéticaRESUMEN
The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume-regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT- and NHEK-LRRC8A knockout cells by using CRISPR-Cas9. I- influx assays using halide-sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein-AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.
Asunto(s)
Aniones/metabolismo , Epidermis/metabolismo , Queratinocitos/citología , Proteínas de la Membrana/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Fluoresceínas/química , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Queratinocitos/metabolismo , Osmorregulación , Ósmosis , Presión Osmótica , Multimerización de Proteína , Análisis de Secuencia de ARNRESUMEN
The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Desoxirribonucleasas/metabolismo , ADN/química , Escherichia coli/genéticaRESUMEN
The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , ADN/química , Proteínas Bacterianas/metabolismo , Calcio/metabolismo , ADN/metabolismo , ADN/ultraestructura , Proteínas de Unión al ADN/metabolismo , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Movimiento (Física) , Unión Proteica , Streptococcus agalactiaeRESUMEN
The LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci. Activation of leuO by RcsB-BglJ is independent of the Rcs phosphorelay system. RcsB-BglJ binds to the leuO promoter region and activates one of two leuO promoters mapped in vivo. Moreover, LeuO antagonizes activation of leuO by RcsB-BglJ and acts as negative autoregulator in vivo and in vitro. Further, the H-NS paralogue StpA causes repression of leuO in addition to H-NS. Together, our data suggest a complex arrangement of regulatory elements and they indicate a feedback control mechanism of leuO expression.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas Fimbrias/antagonistas & inhibidores , Proteínas Fimbrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Operón , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética , Activación Transcripcional , Factores de Virulencia/genéticaRESUMEN
Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Operón , ARN Bacteriano/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Escherichia coli K12/química , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , ARN Bacteriano/química , ARN Bacteriano/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transactivadores/química , Factores de Transcripción/químicaRESUMEN
The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X/métodos , Streptococcus agalactiae/metabolismo , ADN/metabolismo , Unión ProteicaRESUMEN
Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Escherichia coli/fisiología , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Secuencias Invertidas Repetidas , Regiones Promotoras Genéticas , Secuencia de Bases , ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Orden Génico , Modelos Biológicos , Datos de Secuencia Molecular , Familia de Multigenes , ARN Bacteriano/biosíntesis , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/inmunología , Proteínas de Escherichia coli/genética , Factores de Transcripción/genética , Bacteriófago lambda/fisiología , Secuencia de Bases , Clonación Molecular , Huella de ADN , ADN Bacteriano/genética , ADN Intergénico/genética , Escherichia coli K12/virología , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
LRP has recently been shown to interact with the regulatory regions of bacterial ribosomal RNA promoters. Here we study details of the LRP-rDNA interaction by gel retardation and high-resolution footprinting techniques. We show that a second regulator for rRNA transcription, H-NS, facilitates the formation of a higher-order LRP-nucleoprotein complex, probably acting transiently as a DNA chaperone. The macromolecular crowding substance ectoine stabilizes the formation of this dynamic complex, while the amino acid leucine, as a metabolic effector, has the opposite effect. DNase I and hydroxyl radical footprint experiments with LRP-DNA complexes reveal a periodic change of the target DNA structure, which implies extensive DNA wrapping reaching into the promoter core region. We show furthermore that LRP binding is able to constrain supercoils, providing a link between DNA topology and regulation. The results support the conclusion that the bacterial DNA-binding protein LRP, assisted by H-NS, forms a repressive nucleoprotein structure involved in regulation of rRNA transcription. The formation of this regulatory structure appears to be directly affected by environmental changes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína Reguladora de Respuesta a la Leucina/metabolismo , Sustancias Macromoleculares/metabolismo , Transcripción Genética , Regulación Alostérica/efectos de los fármacos , Aminoácidos Diaminos/farmacología , Huella de ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , ADN Ribosómico/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas de Escherichia coli/metabolismo , Factor Proteico para Inverción de Estimulación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Radical Hidroxilo , Conformación de Ácido Nucleico/efectos de los fármacos , Nucleoproteínas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Operón de ARNr/efectos de los fármacos , Operón de ARNr/genéticaRESUMEN
Cascade-crRNA complexes mediate the identification of the invading foreign DNA and initiate its neutralization by formation of an R-loop (RNA-induced DNA-loop) at the crRNA-complementary sequence (protospacer). After initial unspecific binding to the double-stranded DNA, Cascade-crRNA complex slides along the DNA to find the protospacer. Once the target site is detected, the crRNA hybridizes to the complementary strand with subsequent displacement of the non-complementary strand to form an R-loop structure. Here, we describe how Cascade-DNA complexes and the Cascade-induced strand separation can be characterized in detail by combining chemical and enzymatic footprint analyses. Selective modification of unpaired thymines by permanganate (KMnO4) and the specific cleavage of single-stranded DNA by Nuclease P1 can be used to probe an R-loop formation by Cascade. Localization of the Cascade-crRNA complex on the DNA can be achieved by an Exonuclease III protection assay.
Asunto(s)
ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Conformación de Ácido Nucleico , ARN/metabolismo , Sitios de Unión , División del ADN/efectos de los fármacos , Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Modelos Moleculares , Permanganato de Potasio/farmacologíaRESUMEN
The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.
Asunto(s)
ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/virología , Ribonucleoproteínas/química , Secuencia de Bases , Sitios de Unión , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Bacteriano/fisiología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiología , Relación Estructura-Actividad , ARN Pequeño no TraducidoRESUMEN
To study the influence of DNA curvature and DNA-binding proteins, which interact with curved DNA on bacterial promoters, we constructed two sets of promoter variants in which a synthetic DNA-bending module was fused at defined distances and angular orientations with respect to the transcription start sites. The distance between the synthetic binding site centre and the transcription start site of the different constructs varied by up to 20 bp, corresponding to almost two complete helical B-DNA turns. The rRNA promoters rrnB P1 and rrnB P2 were selected as target promoters. While in its natural context P1 depends on upstream curved DNA and several transcription factors that bind to this region, promoter P2 is not preceded by curved DNA, nor is it believed to be directly regulated by transcription factors. In vitro transcription measurements of both promoters in the absence of transcription factors varied with the phase of the curved upstream DNA element, underlining the importance of DNA conformation to promoter efficiency. Specific binding of H-NS and LRP to the curved DNA element was demonstrated by gel shift and footprint analysis. Binding affinity was not notably altered for the different distance variants. We demonstrated that the two proteins acted as repressors for both promoters. The extent of H-NS-mediated repression for both promoters did not vary strongly with the phasing of the upstream binding module. In contrast, LRP-dependent repression showed a clear dependence on the angular orientation of the constructs. Phasing-dependent repression is very distinct for P2 but only rudimentary for the P1 promoter.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteína Reguladora de Respuesta a la Leucina/genética , Regiones Promotoras Genéticas , Algoritmos , Huella de ADN , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Plásmidos , ARN Ribosómico/genética , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
The synthesis of ribosomal RNAs in bacteria is tightly coupled to changes in the environment. This rapid adaptation is the result of several intertwined regulatory networks. The two proteins FIS and H-NS have previously been described to act as antagonistic transcription factors for rRNA synthesis. Here we provide evidence for another player, the regulatory protein LRP, which binds with high specificity to all seven Escherichia coli rRNA P1 promoter upstream regions (UAS). Comparison of the binding properties of LRP and H-NS, and characterization of the stabilities of the various complexes formed with the rRNA UAS regions revealed different binding modes. Binding studies with LRP and H-NS in combination demonstrated that the two proteins interacted with obvious synergism. The efficiency of LRP binding to the rRNA regulatory region is modified by the presence of the effector amino acid leucine, as has been shown for several other operons regulated by this transcription factor. The effect of LRP on the binding of RNA polymerase to the rrnB P1 promoter and in vitro transcription experiments indicated that LRP acts as a transcriptional repressor, thus resembling the activity of H-NS described previously. The results show for the first time that LRP binds to the regulatory region of bacterial rRNA promoters, and very likely contributes in combination with H-NS to the control of rRNA synthesis. From the known properties of LRP a mechanism can be inferred that couples rRNA synthesis to changes in nutritional quality.