RESUMEN
Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.
Asunto(s)
Ecosistema , Flujo Génico/genética , Especiación Genética , Fenotipo , Filogenia , Sulfolobus/genética , Secuencia de Bases , Genética de Población , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Recombinación Homóloga/genética , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Federación de Rusia , Especificidad de la Especie , Sulfolobus/clasificaciónRESUMEN
Virus-host interactions are a key factor shaping population dynamics of microbial species. The CRISPR-Cas adaptive immune system confers sequence-specific immunity to viral infection and has the potential to dramatically shape coevolutionary interactions between viruses and their microbial hosts. To assess evolutionary dynamics of CRISPR loci, we have sampled a population of closely related Sulfolobus islandicus strains from Kamchatka, Russia at two time points, 10 years apart. Sequence analysis of the conserved trailer sequences reveals that alleles are reassorted among three CRISPR spacer loci into combinatorial genotypes. Reassortment provides the evolutionary independence of CRISPR loci from one another as demonstrated by the differential change in allele frequencies between two time points. Genome sequences of 12 strains from this population also reveal very recent horizontal gene transfer of novel, divergent cas gene cassettes. The evolutionary independence of CRISPR loci from each other and of the cas genes that control their function are consistent with the evolutionary expectation that reassortment increases the efficiency of adaptation at these loci that are likely under strong selection by lytic viruses.
Asunto(s)
Virus de Archaea/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Transferencia de Gen Horizontal/genética , Sulfolobus/genética , Sulfolobus/virología , ADN Bacteriano/análisis , ADN Bacteriano/genética , Frecuencia de los Genes/genética , Variación Genética , Datos de Secuencia Molecular , Federación de Rusia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Several studies have shown that American genotype dengue 2 viruses (DENV2) have reduced viral fitness in the mosquito vector, Aedes aegypti, compared to other DENV2 genotypes. Diminished replication efficiency or inability to efficiently traverse membrane barriers encompassing organs such as the midgut or salivary glands are considered major factors negatively impacting viral fitness in the mosquito. RESULTS: We analyzed the vector competence of Ae. aegypti for two American DENV2 strains, QR94 and PR159 originating from Mexico and Puerto-Rico, respectively. Both strains infected mosquito midguts following acquisition of infectious bloodmeals. However, DENV2-QR94 and DENV2-PR159 poorly disseminated from the midgut at 7 or 14 days post-bloodmeal (pbm). We detected one virus isolate, EM33, among 31 DENV2-QR94 infected mosquitoes, and one isolate, EM41, among 121 DENV2-PR159 infected mosquitoes, generating high virus titers in mosquito carcasses at 7 days pbm. In oral challenge experiments, EM33 and EM41 showed midgut dissemination rates of 40-50%. Replication efficiency of EM41 in secondary mosquito tissue was similar to that of a dissemination-competent control strain, whereas the replication efficiency of EM33 was significantly lower than that of the control virus. The genome sequence of DENV2-QR94 encoded seven unique amino acids (aa), which were not found in 100 of the most closely related DENV2 strains. EM33 had one additional aa change, E202K, in the E protein. DENV2-PR159 encoded four unique aa residues, one of them E202K, whereas EM41 had two additional aa substitutions, Q77E in the E protein and E93D in NS3. CONCLUSIONS: Our results indicate that the midgut of Ae. aegypti acts as a selective sieve for DENV2 in which genetically distinct, dissemination-competent virus variants are rapidly selected from the viral quasispecies to be transmitted to vertebrates.
Asunto(s)
Aedes/virología , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Mutación , Animales , Virus del Dengue/fisiología , Femenino , Tracto Gastrointestinal/virología , Genoma Viral , Genotipo , Interacciones Huésped-Patógeno , México , Datos de Secuencia Molecular , Puerto Rico , ARN Viral/genética , Selección Genética , Análisis de Secuencia de ADN , Carga Viral , Replicación ViralRESUMEN
The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.
Asunto(s)
Bacterias/aislamiento & purificación , Bacterias/metabolismo , Manantiales de Aguas Termales/microbiología , Bacterias/clasificación , Bacterias/genética , Colorado , ADN Bacteriano/genética , Datos de Secuencia Molecular , Procesos Fototróficos , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. Through population-scale comparative genomics of 7 Sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. The evolutionary independence of each population allowed us to assess genome dynamics over very recent evolutionary time, beginning approximately 910,000 years ago. On this time scale, genome variation largely consists of recent strain-specific integration of mobile elements. Localized sectors of parallel gene loss are identified; however, the balance between the gain and loss of genetic material suggests that S. islandicus genomes acquire material slowly over time, primarily from closely related Sulfolobus species. Examination of the genome dynamics through population genomics in S. islandicus exposes the process of allopatric speciation in thermophilic Archaea and brings us closer to a generalized framework for understanding microbial genome evolution in a spatial context.
Asunto(s)
Evolución Molecular , Especiación Genética , Variación Genética/genética , Genoma Arqueal/genética , Geografía , Sulfolobus/genética , Proteínas Arqueales/genética , Datos de Secuencia Molecular , Sulfolobus/clasificaciónRESUMEN
Viruses are a driving force of microbial evolution. Despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. One of the primary factors that define viral population structure is coevolution with microbial hosts. Experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. The biogeographic distribution of viruses that infect Sulfolobus islandicus is investigated using genome comparisons among four newly identified, integrated, Sulfolobus spindle-shaped viruses and previously sequenced viral strains. Core gene sequences show a biogeographic distribution where viral genomes are specifically associated with each local population. In addition, signatures of host-virus interactions recorded in the sequence-specific CRISPR (clustered regularly interspaced short palindromic repeats) system show that hosts have interacted with viral communities that are more closely related to local viral strains than to foreign ones. Together, both proviral and CRISPR sequences show a clear biogeographic structure for Sulfolobus viral populations. Our findings demonstrate that virus-microbe coevolution must be examined in a spatially explicit framework. The combination of host and virus biogeography suggests a model for viral diversification driven by host immunity and local adaptation.
Asunto(s)
Genoma Arqueal/genética , Provirus/genética , Sulfolobus/genética , Sulfolobus/virología , Análisis por Conglomerados , Evolución Molecular , Geografía , Interacciones Huésped-Patógeno , Filogenia , Homología de SecuenciaRESUMEN
In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.
Asunto(s)
Aedes/fisiología , Aedes/virología , Membrana Basal/metabolismo , Sangre , Virus Chikungunya/fisiología , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Aedes/anatomía & histología , Animales , Membrana Basal/virología , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Colagenasas/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/virología , Tracto Gastrointestinal/anatomía & histología , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/virología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Proteínas Recombinantes/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Regulación hacia ArribaRESUMEN
In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN). As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP) from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency) where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species.
Asunto(s)
Aedes/genética , Sistemas CRISPR-Cas/genética , Genoma de los Insectos , Fiebre Amarilla/genética , Aedes/patogenicidad , Animales , Secuencia de Bases , Humanos , Mutación INDEL , Edición de ARN/genética , Fiebre Amarilla/transmisión , Dedos de Zinc/genéticaRESUMEN
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system is a recently discovered type of adaptive immune defense in bacteria and archaea that functions via directed incorporation of viral and plasmid DNA into host genomes. Here, we introduce a multiscale model of dynamic coevolution between hosts and viruses in an ecological context that incorporates CRISPR immunity principles. We analyze the model to test whether and how CRISPR immunity induces host and viral diversification and the maintenance of many coexisting strains. We show that hosts and viruses coevolve to form highly diverse communities. We observe the punctuated replacement of existent strains, such that populations have very low similarity compared over the long term. However, in the short term, we observe evolutionary dynamics consistent with both incomplete selective sweeps of novel strains (as single strains and coalitions) and the recurrence of previously rare strains. Coalitions of multiple dominant host strains are predicted to arise because host strains can have nearly identical immune phenotypes mediated by CRISPR defense albeit with different genotypes. We close by discussing how our explicit eco-evolutionary model of CRISPR immunity can help guide efforts to understand the drivers of diversity seen in microbial communities where CRISPR systems are active.
Asunto(s)
Archaea/genética , Bacterias/genética , Bacteriófagos/genética , Evolución Biológica , Secuencias Invertidas Repetidas , Archaea/inmunología , Archaea/virología , Bacterias/inmunología , Bacterias/virología , Bacteriófagos/inmunología , Bacteriófagos/fisiología , Simulación por Computador , Interacciones Huésped-Patógeno , Viabilidad Microbiana , Modelos Genéticos , Mutagénesis , Densidad de Población , Selección GenéticaRESUMEN
BACKGROUND: Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats) are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations. METHODOLOGY/PRINCIPAL FINDINGS: We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population. CONCLUSIONS/SIGNIFICANCE: The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.
Asunto(s)
Biodiversidad , ADN Intergénico/genética , Secuencias Invertidas Repetidas , Sulfolobus/genética , Secuencia de Bases , Manantiales de Aguas Termales/microbiología , Datos de Secuencia Molecular , Filogenia , Federación de Rusia , Sulfolobus/clasificación , Sulfolobus/inmunología , Sulfolobus/aislamiento & purificaciónRESUMEN
Thiol-acrylate photopolymers often contain pendant, unreacted thiol groups even following complete reaction of the acrylate functional groups. The results presented herein demonstrate a high throughput method for quantifying pendant thiol group concentrations using FTIR spectra of thiol-acrylate microspot arrays. Using this technique, more than 25% of the original thiol groups were detected as pendant groups in microspots made from monomer solutions containing at least 40 mol % thiol functional groups. Subsequent modification reactions allowed postpolymerization tailoring of the network chemistry. The extent of modification was controlled by the concentration of the pendant thiols (ranging from 0.01 to 0.4M) and the duration of the modification reaction (0-10 min for photocoupling reactions, 0-24 h for Michael-type addition reactions). Further, when photocoupling was used to modify the networks, spatial and temporal control of the light exposure facilitated the formation of chemical patterns on the surface and throughout the material.
Asunto(s)
Acrilatos/química , Materiales Biocompatibles/química , Oligopéptidos/química , Polímeros/química , Compuestos de Sulfhidrilo/química , Animales , Adhesión Celular , Fibroblastos/metabolismo , Ligandos , Luz , Ensayo de Materiales , Ratones , Microquímica/métodos , Células 3T3 NIH , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Crosslinked, degradable networks formed from the photopolymerization of thiol and acrylate monomers are explored as potential biomaterials. The degradation behavior and material properties of these networks are influenced by the molecular weight of the nondegradable thiol-polyacrylate backbone chains that form during photopolymerization. Here, gel permeation chromatography was used to characterize the thiol-polyacrylate backbone chain lengths in degraded thiol-acrylate networks. Increasing thiol functionality from 1 to 4 increased the backbone molecular weight (MÌ(w) = 2.3 ± 0.07 × 10(4) Da for monothiol and 3.6 ± 0.1 × 10(4) Da for tetrathiol networks). Decreasing thiol functional group concentration from 30 to 10 mol% also increased the backbone lengths (MÌ(w) = 7.3 ± 1.1 × 10(4) Da for the networks containing 10 mol% thiol groups as compared to 3.6 ± 0.1 × 10(4) Da for 30 mol% thiol). Finally, the backbone chain lengths were probed at various stages of degradation and an increase in backbone molecular weight was observed as mass loss progressed from 10 to 70%.