RESUMEN
ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.
Asunto(s)
Bacteriófagos/fisiología , Interacciones Microbiota-Huesped/fisiología , Fosfatos de Inositol/metabolismo , Vibrio cholerae/virología , Proteínas de la Cola de los Virus/metabolismo , Adhesinas Bacterianas , Alelos , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Bacteriófagos/genética , Proteínas de la Cápside/genética , Cólera , Interacciones Microbiota-Huesped/genética , Especificidad del Huésped , Humanos , Fosfatos de Inositol/química , Fosfatos de Inositol/genética , Modelos Animales , Mutación , Mutación Missense , Fenotipo , Porinas/química , Porinas/genética , Porinas/metabolismo , Conejos , Vibrio cholerae/genética , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/genéticaRESUMEN
Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.
Asunto(s)
Interacciones Huésped-Patógeno , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Neumonía Bacteriana/inmunología , Factores de Virulencia/metabolismo , Animales , Elementos Transponibles de ADN , Modelos Animales de Enfermedad , Pruebas Genéticas , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Ratones , Mutagénesis Insercional , Neumonía Bacteriana/microbiología , Virulencia , Factores de Virulencia/genéticaRESUMEN
Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.
Asunto(s)
Acinetobacter baumannii/enzimología , Acinetobacter baumannii/fisiología , Biopelículas/crecimiento & desarrollo , Locomoción , Proteasa La/metabolismo , Viabilidad Microbiana , Estrés FisiológicoRESUMEN
Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.
Asunto(s)
Bacteriófagos/genética , Bacteriófagos/inmunología , Genes Virales/genética , Inmunidad Innata , Vibrio cholerae/inmunología , Vibrio cholerae/virología , Secuencia de Aminoácidos , Bacteriólisis , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/patogenicidad , Secuencia de Bases , Evolución Biológica , Cromosomas Bacterianos/genética , Eliminación de Gen , Genes Virales/inmunología , Genoma Viral/genética , Islas Genómicas/genética , Secuencias Invertidas Repetidas/genética , Datos de Secuencia Molecular , Especificidad por Sustrato , Vibrio cholerae/genéticaRESUMEN
Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate.IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Bacteriano/biosíntesis , Esporas Bacterianas/fisiología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Etanol/farmacología , Genoma Bacteriano , Calor , Mutación , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Radioisótopos de Fósforo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/efectos de la radiaciónRESUMEN
An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Vacunas contra el Cólera/inmunología , Cólera/inmunología , Cólera/microbiología , Antígenos O/inmunología , Vibrio cholerae/inmunología , Animales , Femenino , Inmunoglobulina G/inmunología , Intestino Delgado/microbiología , Ratones , Ratones Endogámicos BALB CRESUMEN
Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection. We performed genetic fitness analyses to identify genes encoding factors contributing to growth on glucose, maltose, mannose, trehalose and N-acetyl-glucosamine. We obtained insight into the potential functions of proteins predicted to be involved in carbohydrate utilization and identified additional factors previously unrecognized as contributing to the metabolism of the tested carbohydrates. Strong phenotypes were observed for the putative carbohydrate phosphotransferase transporters BB0408 and BBB29 as well as the response regulator Rrp1. We further validated Tn-seq for use in mouse studies and were able to correctly identify known infectivity factors as well as additional transporters and genes on lp54 that may contribute to optimal mouse infection. As such, this study establishes Tn-seq as a powerful method for both in vitro and in vivo studies of B. burgdorferi.
Asunto(s)
Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidad , Enfermedad de Lyme/microbiología , Animales , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/genética , Metabolismo de los Hidratos de Carbono/genética , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Aptitud Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Garrapatas/microbiología , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. RESULTS: In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. CONCLUSIONS: We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.
Asunto(s)
Inmunoprecipitación de Cromatina , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación de Cromatina/métodos , Mapeo Cromosómico , Genoma , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
Carbon catabolite repression (CCR) is a regulatory phenomenon implemented by bacteria to hierarchically organize carbohydrate utilization in order to achieve maximal growth. CCR is likely of great importance to Streptococcus pneumoniae because the human host sites inhabited by this pathogen represent complex carbohydrate environments. In this species, inactivation of the prototypical Gram-positive CCR master regulator, ccpA, attenuates virulence in mice but does not relieve CCR of most metabolic enzymes, suggesting CcpA-independent CCR mechanisms predominate. Here we show the activities of three transcriptional regulators constitute the majority of transcriptional CCR of galactose metabolism operons. We determined seryl-phosphorylated histidine phosphocarrier protein (HPr-Serâ¼P)-mediated regulation is a major CCR mechanism and an essential activity in the pneumococcus, as an HPr point mutation abolishing HPrK/P-dependent phosphorylation was not tolerated nor was deletion of hprk/p. The HPr-Serâ¼P phosphomimetic mutant HPr S46D had reduced phosphotransferase system transport rates and limited induction of CCR-repressed genes. These results support a model of pneumococcal CCR in which HPr-Serâ¼P directly affects the activity of CcpA while indirectly affecting the activity of pathway-specific transactional regulators. This report describes the first CcpA-independent CCR mechanism identified in the pneumococcus and the first example of lethality from loss of HPr-Serâ¼P-mediated CCR in any species.
Asunto(s)
Proteínas Bacterianas/metabolismo , Represión Catabólica , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosfoserina/metabolismo , Streptococcus pneumoniae/metabolismo , Animales , Galactosa/metabolismo , Fosforilación , Transporte de Proteínas , Elementos Reguladores de la Transcripción , Proteínas Represoras/metabolismo , Streptococcus pneumoniae/genéticaRESUMEN
Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.
Asunto(s)
Antibacterianos/farmacología , Elementos Transponibles de ADN/genética , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/genética , Ampicilina/farmacología , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Exonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Selección Genética , Vibrio cholerae/genética , Animales , Fusión Artificial Génica , Técnicas Biosensibles , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana , Ratones , Mutagénesis Insercional , Fosfatos/análisis , Activación TranscripcionalRESUMEN
Vibrio cholerae has evolved to adeptly transition between the human small intestine and aquatic environments, leading to water-borne spread and transmission of the lethal diarrheal disease cholera. Using a host model that mimics the pathology of human cholera, we applied high density transposon mutagenesis combined with massively parallel sequencing (Tn-seq) to determine the fitness contribution of >90% of all non-essential genes of V. cholerae both during host infection and dissemination. Targeted mutagenesis and validation of 35 genes confirmed our results for the selective conditions with a total false positive rate of 4%. We identified 165 genes never before implicated for roles in dissemination that reside within pathways controlling many metabolic, catabolic and protective processes, from which a central role for glycogen metabolism was revealed. We additionally identified 76 new pathogenicity factors and 414 putatively essential genes for V. cholerae growth. Our results provide a comprehensive framework for understanding the biology of V. cholerae as it colonizes the small intestine, elicits profuse secretory diarrhea, and disseminates into the aquatic environment.
Asunto(s)
Aptitud Genética , Estadios del Ciclo de Vida/genética , Vibrio cholerae/crecimiento & desarrollo , Vibrio cholerae/genética , Adaptación Fisiológica/genética , Animales , Animales Recién Nacidos , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Viabilidad Microbiana/genética , Organismos Modificados Genéticamente , Conejos , AguaRESUMEN
Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with either protein and should be able to antagonize other anti-phage systems that deplete NAD+.
RESUMEN
The activities of DNA methyltransferases are important for a variety of cellular functions in bacteria. In this study, we developed a modified high-throughput technique called methyl homopolymer tail mediated sequencing (methyl HTM-seq) to identify the undermethylated sites in the Vibrio cholerae genome for the two DNA methyltransferases, Dam, an adenine methyltransferase, and VchM, a cytosine methyltransferase, during growth in rich medium in vitro. Many of the undermethylated sites occurred in intergenic regions, and for most of these sites, we identified the transcription factors responsible for undermethylation. This confirmed the presence of previously hypothesized DNA-protein interactions for these transcription factors and provided insight into the biological state of these cells during growth in vitro. DNA adenine methylation has previously been shown to mediate heritable epigenetic switches in gene regulation. However, none of the undermethylated Dam sites tested showed evidence of regulation by this mechanism. This study is the first to identify undermethylated adenines and cytosines genomewide in a bacterium using second-generation sequencing technology.
Asunto(s)
Metiltransferasas/metabolismo , Vibrio cholerae/enzimología , Metilación de ADN/genética , Metilación de ADN/fisiología , Ensayo de Cambio de Movilidad Electroforética , Metiltransferasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vibrio cholerae/genéticaRESUMEN
Borrelia burgdorferi is an invasive spirochete that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Little is understood about where the bacteria encounter the strongest barriers to infection and how different components of the host immune system influence the population as the infection progresses. To identify population bottlenecks in a murine host, we utilized Tn-seq to monitor the composition of mixed populations of B. burgdorferi during infection. Both wild-type mice and mice lacking the Toll-like receptor adapter molecule MyD88 were infected with a pool of infectious B. burgdorferi transposon mutants with insertions in the same gene. At multiple time points postinfection, bacteria were isolated from the mice and the compositions of the B. burgdorferi populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88(-/-) mice, indicating a role for innate immunity in limiting early establishment of B. burgdorferi infection. There is not a significant bottleneck during the colonization of distal tissues, suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. These findings further our understanding of the interactions between B. burgdorferi and its murine host in the establishment of infection and dissemination of the organism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/patogenicidad , Proteínas Portadoras/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Lipoproteínas/metabolismo , Enfermedad de Lyme/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Borrelia burgdorferi/inmunología , Proteínas Portadoras/genética , Medios de Cultivo/metabolismo , Femenino , Interacciones Huésped-Patógeno , Inmunidad Innata , Lipoproteínas/genética , Enfermedad de Lyme/microbiología , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Mutagénesis Insercional , Factor 88 de Diferenciación Mieloide/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Piel/inmunología , Piel/microbiologíaRESUMEN
BACKGROUND: The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups. RESULTS: Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes. CONCLUSIONS: Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.
Asunto(s)
Técnicas de Genotipaje/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Levaduras/genética , Aneuploidia , Código de Barras del ADN Taxonómico , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Biblioteca de Genes , Recombinación Genética/genética , Levaduras/clasificaciónRESUMEN
BACKGROUND: Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. RESULTS: In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups 'D' (cell division), 'I' (lipid transport and metabolism) and 'J' (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. CONCLUSIONS: A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies.
Asunto(s)
Biblioteca de Genes , Genes Esenciales/genética , Porphyromonas gingivalis/genética , Secuencia de Bases , Mapeo Cromosómico , Biología Computacional , Cartilla de ADN/genética , Elementos Transponibles de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Mutagénesis/genética , Reacción en Cadena de la PolimerasaRESUMEN
The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5'-TT-3' PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5' position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5'-RTT-3' PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5'-RTT-3' PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5' spacer pyrimidine.IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Intergénico/genética , Vibrio cholerae/genética , ADN Bacteriano/genéticaRESUMEN
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.
RESUMEN
A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.