RESUMEN
CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.
Asunto(s)
Bacteriófagos , Proteínas Asociadas a CRISPR , Animales , Bacteriófagos/genética , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , Leptotrichia/genética , Mamíferos/metabolismo , Profagos/genética , Profagos/metabolismo , Ribonucleasas/metabolismoRESUMEN
All immune systems use precise target recognition to interrogate foreign invaders. During CRISPR-Cas immunity, prokaryotes capture short spacer sequences from infecting viruses and insert them into the CRISPR array. Transcription and processing of the CRISPR locus generate small RNAs containing the spacer and repeat sequences that guide Cas nucleases to cleave a complementary protospacer in the invading nucleic acids. In most CRISPR systems, sequences flanking the protospacer drastically affect cleavage. Here, we investigated the target requirements of the recently discovered RNA-targeting type VI-A CRISPR-Cas system in its natural host, Listeria seeligeri. We discovered that target RNAs with extended complementarity between the protospacer flanking sequence and the repeat sequence of the guide RNA are not cleaved by the type VI-A nuclease Cas13, neither in vivo nor in vitro. These findings establish fundamental rules for the design of Cas13-based technologies and provide a mechanism for preventing self-targeting in type VI-A systems.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/genética , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Escherichia coli/genética , Listeria/genética , Ácidos Nucleicos/genética , Ribonucleasas/genéticaRESUMEN
Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers1-3. These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids4,5. In contrast to most Cas nucleases, which destroy invader DNA4-7, the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis- and non-specific trans-RNA cleavage8. Although it has been hypothesized that Cas13 naturally defends against RNA phages8, type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages9,10, suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems11-13, are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.
Asunto(s)
Bacteriófagos/inmunología , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/inmunología , Endodesoxirribonucleasas/metabolismo , Listeria/inmunología , Listeria/virología , Bacteriófagos/genética , Bacteriófagos/crecimiento & desarrollo , Sistemas CRISPR-Cas/genética , Virus ADN/genética , Virus ADN/crecimiento & desarrollo , Virus ADN/inmunología , Listeria/genética , Listeria/crecimiento & desarrollo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.
Asunto(s)
Cristalografía por Rayos X/métodos , Nucleotidiltransferasas/química , Peptidoglicano/metabolismo , Thermus thermophilus/enzimología , Bacillus subtilis/genética , Biocatálisis , Pared Celular/enzimología , Pared Celular/metabolismo , Escherichia coli/genética , Modelos Moleculares , Nucleotidiltransferasas/metabolismo , Dominios Proteicos , Pliegue de Proteína , Relación Estructura-Actividad , Thermus thermophilus/genéticaRESUMEN
Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano/biosíntesis , Polimerizacion , Antibacterianos/farmacología , Bacillus subtilis/citología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , División Celular , Pared Celular/química , Diseño de Fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Mutación , Oligosacáridos/farmacología , Proteínas de Unión a las Penicilinas/clasificación , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano Glicosiltransferasa/química , Peptidoglicano Glicosiltransferasa/genética , FenotipoRESUMEN
When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore's proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities.IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Hidrolasas/genética , Esporas Bacterianas , Proteínas Bacterianas/metabolismo , Hidrolasas/metabolismoRESUMEN
One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Membrana Celular/genética , Proteínas de la Membrana/genética , Ácidos Picolínicos/metabolismo , Esporas Bacterianas/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Transporte Biológico/genética , Membrana Celular/enzimología , Desecación , Calor , Proteínas de la Membrana/metabolismo , Esporas Bacterianas/enzimologíaRESUMEN
The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Comunicación Celular , Técnicas Citológicas , Elementos Transponibles de ADN , Ensayos Analíticos de Alto Rendimiento , Mutación , Esporas BacterianasRESUMEN
During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients, these receptors trigger germination and outgrowth. In a transposon-sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of > 25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger the activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild-type sporulating cells trigger premature germination during differentiation in a GerA-dependent manner. This phenomenon was observed in domesticated and undomesticated wild-type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability and also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Esporas Bacterianas/metabolismo , Alanina , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Genotipo , Proteínas de la Membrana/genética , Operón/genética , Fenotipo , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Pared Celular/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Morfogénesis/fisiología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Cromatografía Líquida de Alta Presión , Microscopía Fluorescente , Filogenia , Plásmidos/genética , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. An early step in the assembly of this transenvelope complex (called the A-Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell-cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A-Q complex and a hub for the localization of mother cell and forespore proteins.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Lipoproteínas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Lipoproteínas/genética , Proteínas de la Membrana/metabolismo , Unión Proteica , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismoRESUMEN
The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both D-amino acids and D-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged D-amino acids. We exploited these observations to design a fluorescent D-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.
Asunto(s)
Pared Celular/metabolismo , Colorantes Fluorescentes/metabolismo , Peptidoglicano/metabolismo , Bacillus subtilis/citología , Bacillus subtilis/enzimología , Escherichia coli/enzimología , Proteínas de Unión a las Penicilinas/metabolismo , Peptidil Transferasas/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismoRESUMEN
Bacteria have evolved anti-viral defenses, but the mechanisms of sensing and stopping infection are still under investigation. In this issue of Cell Host & Microbe, Mets, Kurata, Ernits et al. describe how direct sensing of a phage protein by a bacterial toxin-antitoxin-associated chaperone unleashes toxin activity to prevent infection.
Asunto(s)
Bacteriófagos , Chaperonas Moleculares , Chaperonas Moleculares/metabolismo , Bacteriófagos/fisiología , Sistemas Toxina-Antitoxina , Toxinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Bacterias/virología , Bacterias/metabolismo , Bacterias/genéticaRESUMEN
Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Sistemas CRISPR-Cas , Bacterias/genética , Enzimas de Restricción-Modificación del ADN/genética , ARN Viral/genética , ADNRESUMEN
CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.
Asunto(s)
Bacteriófago lambda/genética , Sistemas CRISPR-Cas , Escherichia coli/genética , Mutación , Recombinación Genética , Bacteriófago lambda/inmunología , Bacteriófago lambda/fisiología , Escherichia coli/inmunología , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Exodesoxirribonucleasa V/genética , Exodesoxirribonucleasa V/inmunología , Interacciones Huésped-Patógeno , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ÏLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.