RESUMEN
Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we revealed that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Lactococcus lactis , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transformación Bacteriana/genética , Lactococcus/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Competencia de la Transformación por ADN/genéticaRESUMEN
Nowadays, the growing human population exacerbates the need for sustainable resources. Inspiration and achievements in nutrient production or human/animal health might emanate from microorganisms and their adaptive strategies. Here, we exemplify the benefits of lactic acid bacteria (LAB) for numerous biotechnological applications and showcase their natural transformability as a fast and robust method to hereditarily influence their phenotype/traits in fundamental and applied research contexts. We described the biogenesis of the transformation machinery and we analyzed the genome of hundreds of LAB strains exploitable for human needs to predict their transformation capabilities. Finally, we provide a stepwise rational path to stimulate and optimize natural transformation with standard and synthetic biology techniques. A comprehensive understanding of the molecular mechanisms driving natural transformation will facilitate and accelerate the improvement of bacteria with properties that serve broad societal interests.
Asunto(s)
Lactobacillales , Animales , Humanos , Lactobacillales/genética , Lactobacillus/genéticaRESUMEN
Almost a century has elapsed since the discovery of bacteriophages (phages), and 85 years have passed since the emergence of evidence that phages can infect starter cultures, thereby impacting dairy fermentations. Soon afterward, research efforts were undertaken to investigate phage interactions regarding starter strains. Investigations into phage biology and morphology and phage-host relationships have been aimed at mitigating the negative impact phages have on the fermented dairy industry. From the viewpoint of a supplier of dairy starter cultures, this review examines the composition of an industrial phage collection, providing insight into the development of starter strains and cultures and the evolution of phages in the industry. Research advances in the diversity of phages and structural bases for phage-host recognition and an overview of the perpetual arms race between phage virulence and host defense are presented, with a perspective toward the development of improved phage-resistant starter culture systems.
Asunto(s)
Interacciones Microbiota-Huesped/fisiología , Lactococcus/virología , Fagos de Streptococcus/fisiología , Industria Lechera , Fagos de Streptococcus/patogenicidadRESUMEN
Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.
Asunto(s)
Siphoviridae/clasificación , Fagos de Streptococcus/clasificación , Streptococcus thermophilus/virología , Microscopía Electrónica de Transmisión , Análisis de Secuencia de ADN , Siphoviridae/genética , Siphoviridae/ultraestructura , Fagos de Streptococcus/genética , Fagos de Streptococcus/ultraestructuraRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phage-host interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not affected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sfi21dt1virus genus) phages but replication of pac-type (Sfi11virus genus) phages was not affected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.
Asunto(s)
Aminopeptidasas/genética , Mutación , Fagos de Streptococcus/fisiología , Streptococcus thermophilus/virología , Aminopeptidasas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Microbiología de Alimentos , Fagos de Streptococcus/genética , Streptococcus thermophilus/enzimología , Streptococcus thermophilus/genética , Replicación Viral , Secuenciación Completa del GenomaRESUMEN
CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.
Asunto(s)
Proteína 9 Asociada a CRISPR/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Bacteriófagos/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Edición Génica , Humanos , Virulencia/genética , Virulencia/fisiologíaRESUMEN
The CRISPR-Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR-Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4-8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.
Asunto(s)
Sistemas CRISPR-Cas/efectos de los fármacos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/efectos de los fármacos , Fagos de Streptococcus/genética , Fagos de Streptococcus/metabolismo , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/virología , Proteínas Virales/genética , Proteínas Virales/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/virología , Edición Génica , Islas Genómicas/genética , Inmunidad , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fenotipo , Mutación Puntual , Profagos , Streptococcus pyogenes/inmunología , Streptococcus thermophilus/genética , Streptococcus thermophilus/virología , Transformación Bacteriana , Proteínas Virales/inmunologíaRESUMEN
Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.
RESUMEN
CRISPR (clustered regularly interspaced short palindromic repeats) together with CAS (RISPR-associated) genes form the CRISPR-Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel 'spacers' into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR-Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.
Asunto(s)
Bacteriófagos/inmunología , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , Genoma Bacteriano/genética , Streptococcus thermophilus/genética , Streptococcus thermophilus/inmunología , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Datos de Secuencia Molecular , Mutación/genéticaRESUMEN
Interactions between bacteria and their coexisting phage populations impact evolution and can strongly influence biogeochemical processes in natural ecosystems. Periodically, mutation or migration results in exposure of a host to a phage to which it has no immunity; alternatively, a phage may be exposed to a host it cannot infect. To explore the processes by which coexisting, co-evolving hosts and phage populations establish, we cultured Streptococcus thermophilus DGCC7710 with phage 2972 and tracked CRISPR (clustered regularly interspaced short palindromic repeats) diversification and host-phage co-evolution in a population derived from a colony that acquired initial CRISPR-encoded immunity. After 1 week of co-culturing, the coexisting host-phage populations were metagenomically characterized using 454 FLX Titanium sequencing. The evolved genomes were compared with reference genomes to identify newly incorporated spacers in S. thermophilus DGCC7710 and recently acquired single-nucleotide polymorphisms (SNPs) in phage 2972. Following phage exposure, acquisition of immune elements (spacers) led to a genetically diverse population with multiple subdominant strain lineages. Phage mutations that circumvented three early immunization events were localized in the proto-spacer adjacent motif (PAM) or near the PAM end of the proto-spacer, suggesting a strong selective advantage for the phage that mutated in this region. The sequential fixation or near fixation of these single mutations indicates selection events so severe that single phage genotypes ultimately gave rise to all surviving lineages and potentially carried traits unrelated to immunity to fixation.
Asunto(s)
Bacteriófagos/fisiología , Mutación , Streptococcus thermophilus/genética , Streptococcus thermophilus/virología , Bacteriófagos/genética , Secuencia de Bases , Evolución Biológica , ADN Intergénico/genética , Variación Genética , Genoma Viral/genética , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido SimpleRESUMEN
The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófagos/fisiología , Secuencias Invertidas Repetidas/genética , Proteómica , Streptococcus thermophilus/genética , Streptococcus thermophilus/virología , Transcriptoma/genética , Sitios Genéticos/genética , Streptococcus thermophilus/citología , Factores de TiempoRESUMEN
BACKGROUND: In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. RESULTS: In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. CONCLUSION: We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácido Láctico/metabolismo , Leche/metabolismo , Leche/microbiología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Animales , Bovinos , Productos Lácteos , Fermentación , Islas Genómicas , Humanos , Fenotipo , Transformación GenéticaRESUMEN
The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , Escherichia coli/genética , Streptococcus thermophilus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/inmunología , Escherichia coli/virología , Inmunidad , Datos de Secuencia Molecular , Mutación , Motivos de Nucleótidos , Plásmidos/genética , Estructura Terciaria de Proteína , Secuencias Repetitivas de Ácidos Nucleicos , Transformación BacterianaRESUMEN
Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Streptococcus thermophilus/enzimología , Adenosina Trifosfato/metabolismo , Clonación Molecular , ADN Helicasas/genética , Análisis Mutacional de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Análisis de Secuencia de ADN , Streptococcus thermophilus/genéticaRESUMEN
Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
Asunto(s)
Bacteriófagos/genética , ADN Viral/metabolismo , Sitios Genéticos/genética , Sitios Genéticos/inmunología , Plásmidos/metabolismo , Streptococcus thermophilus/inmunología , Streptococcus thermophilus/virología , Bacteriófagos/metabolismo , Secuencia de Bases , ADN Intergénico/genética , ADN Intergénico/metabolismo , ADN Viral/genética , Farmacorresistencia Bacteriana/genética , Secuencias Repetitivas Esparcidas/genética , Datos de Secuencia Molecular , Mutación , Plásmidos/genética , ARN Bacteriano/genética , ARN Bacteriano/inmunología , Streptococcus thermophilus/genéticaRESUMEN
A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.
Asunto(s)
Marcación de Gen/métodos , Genética Microbiana/métodos , Streptococcus thermophilus/genética , Mutagénesis Insercional , Eliminación de Secuencia , Transformación BacterianaRESUMEN
In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Feromonas/metabolismo , Percepción de Quorum , Transducción de Señal , Streptococcus thermophilus/fisiología , Transformación Genética , Proteínas Bacterianas/biosíntesis , Perfilación de la Expresión Génica , Operón , Péptidos/genética , Péptidos/metabolismo , Feromonas/genética , Eliminación de Secuencia , Factores de Transcripción/biosíntesisRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in bacteria and archaea, that provide acquired immunity against foreign genetic elements. Here, we investigate the occurrence of CRISPR loci in the genomes of lactic acid bacteria (LAB), including members of the Firmicutes and Actinobacteria phyla. A total of 102 complete and draft genomes across 11 genera were studied and 66 CRISPR loci were identified in 26 species. We provide a comparative analysis of the CRISPR/cas content and diversity across LAB genera and species for 37 sets of CRISPR loci. We analyzed CRISPR repeats, CRISPR spacers, leader sequences, and cas gene content, sequences and architecture. Interestingly, multiple CRISPR families were identified within Bifidobacterium, Lactobacillus and Streptococcus, and similar CRISPR loci were found in distant organisms. Overall, eight distinct CRISPR families were identified consistently across CRISPR repeats, cas gene content and architecture, and sequences of the universal cas1 gene. Since the clustering of the CRISPR families does not correlate with the classical phylogenetic tree, we hypothesize that CRISPR loci have been subjected to horizontal gene transfer and further evolved independently in select lineages, in part due to selective pressure resulting from phage predation. Globally, we provide additional insights into the origin and evolution of CRISPR loci and discuss their contribution to microbial adaptation.
Asunto(s)
Evolución Molecular , Genes Bacterianos , Genoma Bacteriano , Bacterias Grampositivas/genética , Secuencias Invertidas Repetidas/genética , Lactobacillaceae/genética , Regiones no Traducidas 5' , ADN IntergénicoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.