Enrichment and characterization of a nitric oxide-reducing microbial community in a continuous bioreactor.

Nitric oxide (NO) is a highly reactive and climate-active molecule and a key intermediate in the microbial nitrogen cycle. Despite its role in the evolution of denitrification and aerobic respiration, high redox potential and capacity to sustain microbial growth, our understanding of NO-reducing microorganisms remains limited due to the absence of NO-reducing microbial cultures obtained directly from the environment using NO as a substrate. Here, using a continuous bioreactor and a constant supply of NO as the sole electron acceptor, we enriched and characterized a microbial community dominated by two previously unknown microorganisms that grow at nanomolar NO concentrations and survive high amounts (>6 µM) of this toxic gas, reducing it to N2 with little to non-detectable production of the greenhouse gas nitrous oxide. These results provide insight into the physiology of NO-reducing microorganisms, which have pivotal roles in the control of climate-active gases, waste removal, and evolution of nitrate and oxygen respiration.

Unexpected myriad of co-occurring viral strains and species in one of the most abundant and microdiverse viruses on Earth.

Viral genetic microdiversity drives adaptation, pathogenicity, and speciation and has critical consequences for the viral-host arms race occurring at the strain and species levels, which ultimately impact microbial community structure and biogeochemical cycles. Despite the fact that most efforts have focused on viral macrodiversity, little is known about the microdiversity of ecologically important viruses on Earth. Recently, single-virus genomics discovered the putatively most abundant ocean virus in temperate and tropical waters: the uncultured dsDNA virus vSAG 37-F6 infecting Pelagibacter, the most abundant marine bacteria. In this study, we report the cooccurrence of up to ≈1,500 different viral strains (>95% nucleotide identity) and ≈30 related species (80-95% nucleotide identity) in a single oceanic sample. Viral microdiversity was maintained over space and time, and most alleles were the result of synonymous mutations without any apparent adaptive benefits to cope with host translation codon bias and efficiency. Gene flow analysis used to delimitate species according to the biological species concept (BSC) revealed the impact of recombination in shaping vSAG 37-F6 virus and Pelagibacter speciation. Data demonstrated that this large viral microdiversity somehow mirrors the host species diversity since ≈50% of the 926 analyzed Pelagibacter genomes were found to belong to independent BSC species that do not significantly engage in gene flow with one another. The host range of this evolutionarily successful virus revealed that a single viral species can infect multiple Pelagibacter BSC species, indicating that this virus crosses not only formal BSC barriers but also biomes since viral ancestors are found in freshwater.

Minimal-moderate variation of human oral virome and microbiome in IgA deficiency.

Immunoglobulin A (IgA) is the dominant antibody found in our mucosal secretions and has long been recognized to play an important role in protecting our epithelium from pathogens. Recently, IgA has been shown to be involved in gut homeostatic regulation by 'recognizing' and shaping our commensal microbes. Paradoxically, yet selective IgA-deficiency is often described as asymptomatic and there is a paucity of studies only focused on the mice and human gut microbiome context fully ignoring other niches of our body and our commensal viruses. Here, we used as a model the human oral cavity and employed a holistic view and studied the impact of IgA deficiency and also common variable IgA and IgM immunodeficiencies (CVID), on both the human virome and microbiome. Unexpectedly, metagenomic and experimental data in human IgA deficiency and CVID indicate minimal-moderate changes in microbiome and virome composition compared to healthy control group and point out to a rather functional, resilient oral commensal viruses and microbes. However, a significant depletion (two fold) of bacterial cells (p-value < 0.01) and viruses was observed in IgA-deficiency. Our results demonstrate that, within the limits of our cohort, IgA role is not critical for maintaining a rather functional salivary microbiome and suggest that IgA is not a major influence on the composition of abundant commensal microbes.

Benchmarking of single-virus genomics: a new tool for uncovering the virosphere.

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome.

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2-79.1%) and Janthinobacterium (0.7-13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter.

Wastewater treatment plants effluents are considered as hotspots for the dispersion of antibiotic resistance genes (ARGs) into natural ecosystems. The bacterial resistome (ARG collection in a metagenome) analyses have provided clues on antibacterial resistance dynamics. However, viruses and vesicles are frequently ignored. Here, we addressed the bacterial, viral and vesicle resistomes from a representative wastewater effluent in natural conditions and amended with polymyxin, which is used as a last resort antibiotic. Metagenomics showed that the natural prokaryotic resistome was vast (9000 ARG hits/Gb metagenome) and diverse, while viral resistome was two orders of magnitude lower (50 ARG hits/Gb metagenome) suggesting that viruses rarely encoded ARGs. After polymyxin amendment, data showed no ARG enrichment - including to polymyxin - in the microbiome. Remarkably, microbiomes responded to polymyxin with a vast release of putative vesicles (threefold increase compared with the control), which might be used as 'traps' to decrease the antibiotic concentration. Intriguingly, although polymyxin resistance genes (PRGs) were rare in the microbiome (0.018% of total ARG found), in the viral and vesicle fractions, PRGs were more abundant (0.5%-0.8% of total ARG found). Our data suggest that vesicles could have a more active role in the context of transmission of antibiotic resistances.

Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses.

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.

Airway microbiota in patients with paediatric cystic fibrosis: Relationship with clinical status.

INTRODUCTION: New massive sequencing techniques make it possible to determine the composition of airway microbiota in patients with cystic fibrosis (CF). However, the relationship between the composition of lung microbiome and the clinical status of paediatric patients is still not fully understood. MATERIAL AND METHODS: A cross-sectional observational study was conducted on induced sputum samples from children with CF and known mutation in the CFTR gene. The bacterial sequences of the 16SrRNA gene were analyzed and their association with various clinical variables studied. RESULTS: Analysis of the 13 samples obtained showed a core microbiome made up of Staphylococcus spp., Streptococcus spp., Rothia spp., Gemella spp. and Granulicatella spp., with a small number of Pseudomonas spp. The cluster of patients with less biodiversity were found to exhibit a greater number of sequences of Staphylococcus spp., mainly Staphylococcus aureus (p 0.009) and a greater degree of lung damage. CONCLUSION: An airway microbiome with greater biodiversity may be an indicator of less pronounced disease progression, in which case new therapeutic interventions that prevent reduction in non-pathogenic species of the airway microbiota should be studied.

Airway microbiota in patients with paediatric cystic fibrosis: Relationship with clinical status / Microbiota respiratoria en pacientes pediátricos con fibrosis quística: relación con la situación clínica

Introduction: New massive sequencing techniques make it possible to determine the composition of airway microbiota in patients with cystic fibrosis (CF). However, the relationship between the composition of lung microbiome and the clinical status of paediatric patients is still not fully understood. Material and methods: A cross-sectional observational study was conducted on induced sputum samples from children with CF and known mutation in the CFTR gene. The bacterial sequences of the 16SrRNA gene were analyzed and their association with various clinical variables studied. Results: Analysis of the 13 samples obtained showed a core microbiome made up of Staphylococcus spp., Streptococcus spp., Rothia spp., Gemella spp. and Granulicatella spp., with a small number of Pseudomonas spp. The cluster of patients with less biodiversity were found to exhibit a greater number of sequences of Staphylococcus spp., mainly Staphylococcus aureus (p 0.009) and a greater degree of lung damage. Conclusion: An airway microbiome with greater biodiversity may be an indicator of less pronounced disease progression, in which case new therapeutic interventions that prevent reduction in non-pathogenic species of the airway microbiota should be studied

Introducción: Las nuevas técnicas de secuenciación masiva permiten determinar la composición de la microbiota de las vías respiratorias en pacientes con fibrosis quística (FQ). Sin embargo, la relación entre la composición de la microbiota pulmonar y el estado clínico de los pacientes pediátricos todavía no se ha establecido bien. Material y métodos: Se realizó un estudio transversal observacional en muestras de esputo inducido de niños con FQ y mutación conocida en el gen CFTR. Se analizaron las secuencias bacterianas del gen 16SrRNA y se estudió su asociación con diversas variables clínicas. Resultados: El análisis de las 13 muestras obtenidas mostró un microbioma central compuesto por Staphylococcus spp., Streptococcus spp., Rothia spp., Gemella spp. y Granulicatella spp., con un pequeño número de Pseudomonas spp. Se descubrió que el grupo de pacientes con menos biodiversidad mostraba un mayor número de secuencias de Staphylococcus spp., principalmente Staphylococcus aureus (p 0,009) y un mayor daño de la función pulmonar. Conclusión: La mayor biodiversidad del microbioma de vía respiratoria puede ser un indicador de menor progresión de la enfermedad, en cuyo caso deben estudiarse nuevas intervenciones terapéuticas que prevengan la disminución de especies no patógenas

Single-cell genomics uncover Pelagibacter as the putative host of the extremely abundant uncultured 37-F6 viral population in the ocean.

The identification of relevant virus-host pairs that globally account for a large pool of carbon and nutrients in the ocean is paramount to build accurate ecological models. A previous work using single-virus genomics led to the discovery of the uncultured single-virus vSAG 37-F6, originally sorted from the Mediterranean Sea (Blanes Bay Microbial Observatory), that represents one of the most abundant dsDNA viral population in the marine surface virosphere. Here, from same sampling site, we report that a Pelagibacter single-cell contained a viral member of vSAG 37-F6 population, by means of PCR screening of sorted, genome-amplified single cells with vSAG 37-F6-specific primers and whole-genome sequencing. Furthermore, viruses from this population were also found in three other Pelagibacter single cells from the South Pacific and Atlantic oceans. These new uncultured pelagiphages were genetically different from the previously characterized pelagiphage isolates. Data showed that the uncultured vSAG 37-F6 population represents the Pelagibacter phages that inhabit the sunlit ocean better, and contains a vast unrecognized microdiversity.

Deciphering the Human Virome with Single-Virus Genomics and Metagenomics.

Single-cell genomics has unveiled the metabolic potential of dominant microbes inhabiting different environments, including the human body. The lack of genomic information for predominant microbes of the human body, such as bacteriophages, hinders our ability to answer fundamental questions about our viral communities. Here, we applied single-virus genomics (SVGs) to natural human salivary samples in combination with viral metagenomics to gain some insights into the viral community structure of the oral cavity. Saliva samples were processed for viral metagenomics (n = 15) and SVGs (n = 3). A total of 1328 uncultured single viruses were sorted by fluorescence-activated virus sorting followed by whole genome amplification. Sequencing of 24 viral single amplified genomes (vSAGs) showed that half of the vSAGs contained viral hallmark genes. Among those bona fide viruses, the uncultured single virus 92-C13 putatively infecting oral Streptococcus-like species was within the top ≈10 most abundant viruses in the oral virome. Viral gene network and viral metagenomics analyses of 439 oral viruses from cultures, metagenomics, and SVGs revealed that salivary viruses were tentatively structured into ≈200 major viral clusters, corresponding to approximately genus-level groupings. Data showed that none of the publicly available viral isolates, excepting an Actinomyces phage, were significantly abundant in the oral viromes. In addition, none of the obtained viral contigs and vSAGs from this study were present in all viromes. Overall, the data demonstrates that most viral isolates are not naturally abundant in saliva, and furthermore, the predominant viruses in the oral cavity are yet uncharacterized. Results suggest a variable, complex, and interpersonal viral profile. Finally, we demonstrated the power of SVGs in combination with viral metagenomics to unveil the genetic information of the uncultured viruses of the human virome.

A new class of marine Euryarchaeota group II from the Mediterranean deep chlorophyll maximum.

We have analyzed metagenomic fosmid clones from the deep chlorophyll maximum (DCM), which, by genomic parameters, correspond to the 16S ribosomal RNA (rRNA)-defined marine Euryarchaeota group IIB (MGIIB). The fosmid collections associated with this group add up to 4 Mb and correspond to at least two species within this group. From the proposed essential genes contained in the collections, we infer that large sections of the conserved regions of the genomes of these microbes have been recovered. The genomes indicate a photoheterotrophic lifestyle, similar to that of the available genome of MGIIA (assembled from an estuarine metagenome in Puget Sound, Washington Pacific coast), with a proton-pumping rhodopsin of the same kind. Several genomic features support an aerobic metabolism with diversified substrate degradation capabilities that include xenobiotics and agar. On the other hand, these MGIIB representatives are non-motile and possess similar genome size to the MGIIA-assembled genome, but with a lower GC content. The large phylogenomic gap with other known archaea indicates that this is a new class of marine Euryarchaeota for which we suggest the name Thalassoarchaea. The analysis of recruitment from available metagenomes indicates that the representatives of group IIB described here are largely found at the DCM (ca. 50 m deep), in which they are abundant (up to 0.5% of the reads), and at the surface mostly during the winter mixing, which explains formerly described 16S rRNA distribution patterns. Their uneven representation in environmental samples that are close in space and time might indicate sporadic blooms.

Novel group of podovirus infecting the marine bacterium Alteromonas macleodii.

Four novel, closely related podoviruses, which displayed lytic activity against the gamma-proteobacterium Alteromonas macleodii, have been isolated and sequenced. Alterophages AltAD45-P1 to P4 were obtained from water recovered near a fish farm in the Mediterranean Sea. Their morphology indicates that they belong to the Podoviridae. Their linear and dsDNA genomes are 100-104 kb in size, remarkably larger than any other described podovirus. The four AltAD45-phages share 99% nucleotide sequence identity over 97% of their ORFs, although an insertion was found in AltAD45-P1 and P2 and some regions were slightly more divergent. Despite the high overall sequence similarity among these four phages, the group with the insertion and the group without it, have different host ranges against the A. macleodii strains tested. The AltAD45-P1 to P4 phages have genes for DNA replication and transcription as well as structural genes, which are similar to the N4-like Podoviridae genus that is widespread in proteobacteria. However, in terms of their genomic structure, AltAD45-P1 to P4 differ from that of the N4-like phages. Some distinguishing features include the lack of a large virion encapsidated RNA polymerase gene, very well conserved among all the previously described N4-like phages, a single-stranded DNA binding protein and different tail protein genes. We conclude that the AltAD45 phages characterized in this study constitute a new genus within the Podoviridae.

Reconstruction of novel cyanobacterial siphovirus genomes from Mediterranean metagenomic fosmids.

Cellular metagenomes are primarily used for investigating microbial community structure and function. However, cloned fosmids from such metagenomes capture phage genome fragments that can be used as a source of phage genomes. We show that fosmid cloning from cellular metagenomes and sequencing at a high coverage is a credible alternative to constructing metaviriomes and allows capturing and assembling novel, complete phage genomes. It is likely that phages recovered from cellular metagenomes are those replicating within cells during sample collection and represent "active" phages, naturally amplifying their genomic DNA and increasing chances for cloning. We describe five sets of siphoviral contigs (MEDS1, MEDS2, MEDS3, MEDS4, and MEDS5), obtained by sequencing fosmids from the cellular metagenome of the deep chlorophyll maximum in the Mediterranean. Three of these represent complete siphoviral genomes and two represent partial ones. This is the first set of phage genomes assembled directly from cellular metagenomic fosmid libraries. They exhibit low sequence similarities to one another and to known siphoviruses but are remarkably similar in overall genome architecture. We present evidence suggesting they infect picocyanobacteria, likely Synechococcus. Four of these sets also define a novel branch in the phylogenetic tree of phage large subunit terminases. Moreover, some of these siphoviral groups are globally distributed and abundant in the oceans, comparable to some known myoviruses and podoviruses. This suggests that, as more siphoviral genomes become available, we will be better able to assess the abundance and influence of this diverse and polyphyletic group in the marine habitat.

Polyclonality of concurrent natural populations of Alteromonas macleodii.

We have analyzed a natural population of the marine bacterium, Alteromonas macleodii, from a single sample of seawater to evaluate the genomic diversity present. We performed full genome sequencing of four isolates and 161 metagenomic fosmid clones, all of which were assigned to A. macleodii by sequence similarity. Out of the four strain genomes, A. macleodii deep ecotype (AltDE1) represented a different genome, whereas AltDE2 and AltDE3 were identical to the previously described AltDE. Although the core genome (~80%) had an average nucleotide identity of 98.51%, both AltDE and AltDE1 contained flexible genomic islands (fGIs), that is, genomic islands present in both genomes in the same genomic context but having different gene content. Some of the fGIs encode cell surface receptors known to be phage recognition targets, such as the O-chain of the lipopolysaccharide, whereas others have genes involved in physiological traits (e.g., nutrient transport, degradation, and metal resistance) denoting microniche specialization. The presence in metagenomic fosmids of genomic fragments differing from the sequenced strain genomes, together with the presence of new fGIs, indicates that there are at least two more A. macleodii clones present. The availability of three or more sequences overlapping the same genomic region also allowed us to estimate the frequency and distribution of recombination events among these different clones, indicating that these clustered near the genomic islands. The results indicate that this natural A. macleodii population has multiple clones with a potential for different phage susceptibility and exploitation of resources, within a seemingly unstructured habitat.

Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses.

BACKGROUND: Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales). CONCLUSIONS AND SIGNIFICANCE: We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.