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Pathogen emergence remains one of the most pressing public health concerns of our times. Here, using the agent of cholera, theVibrio choleraepandemic cholera group (PCG) as a model system, we investigate the evolutionary dynamics that lead to the emergence of human pathogens from environmental populations. Genomic comparison of over 1,100V. choleraegenomes including novel isolates from this study, reveal a generalized cluster-driven phylogeny and evolution of the species. Emergence of PCG is largely based on modular acquisition of mobile genetic elements including small gene clusters and allelic variations, with distinct phylogenomic clusters acting as differential reservoirs of virulence factors. Surprisingly, PCG encodes few unique genes, which are mostly encoded within the super-integron, however, many genes within the group exhibit an aberrant degree of positive selection. Our analyses provide a compelling scenario for the emergence of pandemic clones inV. choleraestablishing a blueprint for other bacterial pathogens.

Background The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former, albeit with lower salinity and temperature. Despite its well-known hydrology and physicochemical features, this enormous water mass remains poorly studied at the microbial genomics level. Results We have sampled its different water masses and analyzed the microbiome by shotgun and genome-resolved metagenomics, generating a large number of metagenome-assembled genomes (MAGs) from them. We found various similarities with previously described Black Sea metagenomic datasets, that show remarkable stability in its microbiome. Our datasets are also comparable to other marine anoxic water columns like the Cariaco Basin. The oxic zone resembles to standard marine (e.g. Mediterranean) photic zones, with Cyanobacteria (Synechococcus but a conspicuously absent Prochlorococcus), and photoheterotrophs domination (largely again with marine relatives). The chemocline presents very different characteristics from the oxic surface with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. The euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy-generating metabolism, a few (but detectable) methanogenesis marker genes, and a large number of “dark matter” streamlined genomes of largely unpredictable ecology. Conclusions The Black Sea oxic zone presents many similarities to the global ocean while the redoxcline and euxinic water masses have similarities to other similar aquatic environments of marine (Cariaco Basin or other Black Sea regions) or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environment. We are adding critical information about this unique and important ecosystem and its microbiome.

The recovery of DNA from viromes is a major obstacle in the use of long-read sequencing to study their genomes. For this reason, the use of cellular metagenomes (0.2-μm size range) emerges as an interesting complementary tool, since they contain large amounts of naturally amplified viral genomes from prelytic replication. We have applied second-generation (Illumina NextSeq; short reads) and third-generation (PacBio Sequel II; long reads) sequencing to compare the diversity and features of the viral community in a marine sample obtained from offshore waters of the western Mediterranean. We found that a major wedge of the expected marine viral diversity was directly recovered by the raw PacBio circular consensus sequencing (CCS) reads. More than 30,000 sequences were detected only in this data set, with no homologues in the long- and short-read assembly, and ca. 26,000 had no homologues in the large data set of the Global Ocean Virome 2 (GOV2), highlighting the information gap created by the assembly bias. At the level of complete viral genomes, the performance was similar in both approaches. However, the hybrid long- and short-read assembly provided the longest average length of the sequences and improved the host assignment. Although no novel major clades of viruses were found, there was an increase in the intraclade genomic diversity recovered by long reads that produced an enriched assessment of the real diversity and allowed the discovery of novel genes with biotechnological potential (e.g., endolysin genes).

Pseudomonas aeruginosa is an opportunistic human pathogen and a major cause of nosocomial infections. The global spread of carbapenem-resistant strains is growing rapidly and has become a major public health challenge. Imipenem-relebactam (I/R) is a novel carbapenem-beta-lactamase inhibitor combination that can overcome carbapenem resistance. In this study, we aimed to understand the mechanism underlying resistance to imipenem and imipenem-relebactam. For this purpose, we performed a genomic comparison of 40 new clinical P. aeruginosa strains with different antibiotic sensitivity patterns as well as the presence/absence of carbapenemases. Results indicated the presence of a reduced flexible genome (15% total) mostly represented by phages and defense mechanisms against them, showing an important role in evolution and pathogenicity. We found a high diversity of antibiotic resistance genes grouped in small clusters mobilized via integrative and conjugative elements and facilitated by the high homologous recombination detected. Ortholog genes were found in several pathogenic strains from distantly related taxa in different mobile elements with a global distribution. The microdiversity found in those strains without carbapenemases did not reveal a clear pattern that could be associated with carbapenem resistance, suggesting multiple mechanisms of resistance in the core genome. Our results provide new insight into the dynamics and high genomic plasticity by which clinical strains of P. aeruginosa acquire resistance. This knowledge can be applied to other multidrug-resistant microbes to create predictive frameworks for assessing common molecular mechanisms of antibiotic resistance and integrated into new strategies for their prevention. IMPORTANCE The growing emergence and spread of carbapenem-resistant pathogens worldwide exacerbate the clinical challenge of treating these infections. Given the importance of carbapenems for the treatment of infections caused by Pseudomonas aeruginosa, this study aimed to investigate the underlying genomic properties of the clinical isolates that exhibited resistance to imipenem and imipenem-relebactam. This information will enhance our ability to forecast traits of resistant strains and design reliable treatments against this important threat. Our results provide new insight into the dynamics and high genomic plasticity by which clinical strains of P. aeruginosa acquire resistance as well as offers a methodology that can be applied to many other opportunistic pathogens with broad antibiotic resistance.

The SAR116 clade within the class Alphaproteobacteria represents one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. The small number of cultured representatives of SAR116 (only two to date) is a major bottleneck that has prevented an in-depth study at the genomic level to understand the relationship between genome diversity and its role in the marine environment. In this study, we use all publicly available genomes to provide a genomic overview of the phylogeny, metabolism, and biogeography within the SAR116 clade. This increased genomic diversity has led to the discovery of two subclades that, despite coexisting in the same environment, display different properties in their genomic makeup. One represents a novel subclade for which no pure cultures have been isolated and is composed mainly of single-amplified genomes (SAGs). Genomes within this subclade showed convergent evolutionary trajectories with more streamlined features, such as low GC content (ca. 30%), short intergenic spacers (

Third-generation sequencing has penetrated little in metagenomics due to the high error rate and dependence for assembly on short-read designed bioinformatics. However, second-generation sequencing metagenomics (mostly Illumina) suffers from limitations, particularly in the assembly of microbes with high microdiversity and retrieval of the flexible (adaptive) fraction of prokaryotic genomes. Here, we have used a third-generation technique to study the metagenome of a well-known marine sample from the mixed epipelagic water column of the winter Mediterranean. We have compared PacBio Sequel II with the classical approach using Illumina Nextseq short reads followed by assembly to study the metagenome. Long reads allow for efficient direct retrieval of complete genes avoiding the bias of the assembly step. Besides, the application of long reads on metagenomic assembly allows for the reconstruction of much more complete metagenome-assembled genomes (MAGs), particularly from microbes with high microdiversity such as Pelagibacterales. The flexible genome of reconstructed MAGs was much more complete containing many adaptive genes (some with biotechnological potential). PacBio Sequel II CCS appears particularly suitable for cellular metagenomics due to its low error rate. For most applications of metagenomics, from community structure analysis to ecosystem functioning, long reads should be applied whenever possible. Specifically, for in silico screening of biotechnologically useful genes, or population genomics, long-read metagenomics appears presently as a very fruitful approach and can be analyzed from raw reads before a computationally demanding (and potentially artifactual) assembly step.

The SAR116 clade within the class Alphaproteobacteria represents one of the most abundant groups of heterotrophic bacteria inhabiting the surface of the ocean. The small number of cultured representatives of SAR116 (only two to date) is a major bottleneck that has prevented an in-depth study at the genomic level to understand the relationship between genome diversity and its role in the marine environment. In this study, we use all publicly available genomes to provide a genomic overview of the phylogeny, metabolism and biogeography within the SAR116 clade. This increased genomic diversity revealed has led to the discovery of two subclades of SAR116 that, despite having similar genome size (ca. 2.4 Mb) and coexist in the same environment, display different properties in their genomic make up. One represents a novel subclade for which no pure cultures have been isolated and is composed mainly of single-amplified genomes (SAGs). Genomes within this subclade showed convergent evolutionary trajectories with more streamlining features, such as low GC content (ca. 30%), short intergenic spacers (N/dS). Besides, they were more abundant in metagenomic databases recruiting also at the deep chlorophyll maximum. Less abundant and restricted to the upper photic layers of the global ocean, the other subclade of SAR116, enriched in MAGs, accommodated the only two pure cultures. Genomic analysis suggested that both clades have a significant role in the sulfur cycle with differences in the way in which both clades can metabolize the dimethylsulfoniopropionate (DMSP).IMPORTANCESAR116 clade of Alphaproteobacteria is an ubiquitous group of heterotrophic bacteria inhabiting the surface of the ocean, but the information about their ecology and population genomic diversity is scarce due to the difficulty of getting pure culture isolates. The combination of single-cell genomics and metagenomics has become an alternative approach to study this kind of microbes. Our results expand the understanding of the genomic diversity, distribution, and lifestyles within this clade and provide evidence of different evolutionary trajectories in the genome make-up of the two subclades that could serve to understand how evolutionary pressure can drive different adaptations to the same environment. Therefore, the SAR116 clade represents an ideal model organism for the study of the evolutionary streamlining of genomes in microbes that have relatively close relatedness to each other.

BackgroundSARS-CoV-2 is an RNA virus causing COVID-19. The clinical characteristics and epidemiology of COVID-19 have been extensively investigated, however studies focused on the patient’s microbiota are still lacking. In this study, we investigated the nasopharyngeal microbiome composition of patients who developed different severity levels of COVID-19. We performed Rdna-SSU (16S) sequencing from nasopharyngeal swab samples obtained from SARS-CoV-2 positive (56) and negative (18) patients in the province of Alicante (Spain) in their first visit to the hospital. Positive SARS-CoV-2 patients were observed and later categorized in mild (symptomatic without hospitalization), moderate (hospitalization) and severe (admission to ICU). We compared the microbiome diversity and OTU composition among severity groups using Similarity Percentage (SIMPER) analysis and Maaslin2. We also built bacterial co-abundance networks for each group using Fastpar.ResultsStatistical analysis indicated differences in the nasopharyngeal microbiome of COVID19 patients. 62 OTUs were found exclusively in SARS-CoV-2 positive patients, mostly classified as members of the phylum Bacteroidetes (18) and Firmicutes (25). OTUs classified as Prevotella were found to be significantly more abundant in patients that developed more severe COVID-19. Furthemore, co-abundance analysis indicated a loss of network complexity among samples from patients that later developed more severe symptoms.ConclusionsOur preliminary study shows that the nasopharyngeal microbiome of COVID-19 patients showed differences in the composition of specific OTUs and complexity of co-abundance networks. These microbes with differential abundances among groups could serve as biomarkers for COVID-19 severity. Nevertheless, further studies with larger sample sizes should be conducted to validate these results.IMPORTANCEThis work has studied the microbiota of the nasopharyngeal tract in COVID19 patients using advanced techniques of molecular microbiology. Diverse microorganisms, most of which are harmless or even beneficial to the host, colonize the nasopharyngeal tract. These microorganisms are the microbiota, and they are present in every people. However, changes in this microbiota could be related to different diseases as cancer, gastrointestinal pathologies or even COVID19. This study has been performed to investigate the microbiota from patients with COVID19, in order to determinate its implication in the pathology severity. The results obtained showed that it is possible that several specific microorganisms are present only in patients with severe COVID19. These data, could be used as a prognostic biomarker to early detect whose patients will develop a severe COVID19 and improve their clinical management.

Microbiology is in a new age in which sequence databases are primary sources of information about many microbes. However, in-depth analysis of environmental genomes thus retrieved is essential to substantiate the new knowledge., “Candidatus Actinomarinales” was defined as a subclass of exclusively marine Actinobacteria with small cells and genomes. We have collected all the available genomes in databases to assess the diversity included in this group and analyzed it by comparative genomics. We have found the equivalent of five genera and 18 genomospecies. They have genome reduction parameters equal to those of freshwater actinobacterial “Candidatus Nanopelagicales” or marine alphaproteobacterial Pelagibacterales. Genome recruitment shows that they are found only in the photic zone and mainly in surface waters, with only one genus that is found preferentially at or below the deep chlorophyll maximum. “Ca. Actinomarinales” show a highly conserved core genome (80% of the gene families conserved for the whole order) with a saturation of genomic diversity of the flexible genome at the genomospecies level. We found only a flexible genomic island preserved throughout the order; it is related to the sugar decoration of the envelope and uses several tRNAs as hot spots to increase its genomic diversity. Populations had a discrete level of sequence diversity similar to other marine microbes but drastically different from the much higher levels found for Pelagibacterales. Genomic analysis suggests that they are all aerobic photoheterotrophs with one type 1 rhodopsin and a heliorhodopsin. Like other actinobacteria, they possess the F420 coenzyme biosynthesis pathway, and its lower reduction potential could provide access to an increased range of redox chemical transformations. Last, sequence analysis revealed the first “Ca. Actinomarinales” phages, including a prophage, with metaviromic islands related to sialic acid cleavage. IMPORTANCE Microbiology is in a new age in which sequence databases are primary sources of information about many microbes. However, in-depth analysis of environmental genomes thus retrieved is essential to substantiate the new knowledge. Here, we study 182 genomes belonging to the only known exclusively marine pelagic group of the phylum Actinobacteria. The aquatic branch of this phylum is largely known from environmental sequencing studies (single-amplified genomes [SAGs] and metagenome-assembled genomes [MAGs]), and we have collected and analyzed the available information present in databases about the “Ca. Actinomarinales.” They are among the most streamlined microbes to live in the epipelagic zone of the ocean, and their study is critical to obtain a proper view of the diversity of Actinobacteria and their role in aquatic ecosystems.

BackgroundThird-generation sequencing has penetrated little in metagenomics due to the high error rate and dependence for assembly on short-read designed bioinformatics. However, 2nd generation sequencing metagenomics (mostly Illumina) suffers from limitations, particularly in allowing assembly of microbes with high microdiversity or retrieving the flexible (adaptive) compartment of prokaryotic genomes.ResultsHere we have used different 3rd generation techniques to study the metagenome of a well-known marine sample from the mixed epipelagic water column of the winter Mediterranean. We have compared Oxford Nanopore and PacBio last generation technologies with the classical approach using Illumina short reads followed by assembly. PacBio Sequel II CCS appears particularly suitable for cellular metagenomics due to its low error rate. Long reads allow efficient direct retrieval of complete genes (473M/Tb) and operons before assembly, facilitating annotation and compensates the limitations of short reads or short-read assemblies. MetaSPAdes was the most appropriate assembly program when used in combination with short reads. The assemblies of the long reads allow also the reconstruction of much more complete metagenome-assembled genomes, even from microbes with high microdiversity. The flexible genome of reconstructed MAGs is much more complete and allows rescuing more adaptive genes.ConclusionsFor most applications of metagenomics, from community structure analysis to ecosystem functioning, long-reads should be applied whenever possible. Particularly for in-silico screening of biotechnologically useful genes, or population genomics, long-read metagenomics appears presently as a very fruitful approach and can be used from raw reads, before a computing-demanding (and potentially artefactual) assembly step.

Background: The Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former albeit with lower salinity and (mostly) temperature. In spite of its well-known hydrology and physico chemistry, this enormous water mass remains poorly studied at the microbial genomics level. Results: We have sampled its different water masses and analyzed the microbiome by classic and genome-resolved metagenomics generating a large number of metagenome-assembled genomes (MAGs) from them. The oxic zone presents many similarities to the global ocean while the euxinic water mass has similarities to other similar aquatic environments of marine or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environments and includes Cyanobacteria (Synechococcus), photoheterotrophs (largely with marine relatives), facultative/microaerophilic microbes again largely marine, chemolithotrophs (N and S oxidizers) and a large number of anaerobes, mostly sulfate reducers but also a few methanogens and a large number of “dark matter” streamlined genomes of largely unpredictable ecology. Conclusions: The Black Sea presents a mixture of similarities to other water bodies. The photic zone has many microbes in common with that of the Mediterranean with the relevant exception of the absence of Prochlorococcus. The chemocline already presents very different characteristics with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. Finally the euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy generating metabolism and a small but detectable methanogenesis.We are adding critical information about this unique and important ecosystem and its microbiome.

The increasing demand for products for human consumption is leading to the fast-growing expansion of numerous food sectors such as marine aquaculture (mariculture). However, excessive input of nutrients and pollutants modifies marine ecosystems. Here, we applied a metagenomic approach to investigate these perturbations in samples from marine farms of gilthead seabream cultures. Results revealed dysbiosis and functional imbalance within the net cage with a unique structure, with little interference with samples from the fish microbiota or those collected far away from the coast. Remarkably, below the cage the prokaryotic community was highly similar to the marine microbiome of photic offshore samples. We recovered 48 novel metagenome-assembled genomes. Metagenomic recruitment revealed a significant change in the microbial community which was dominated by several Proteobacteria orders (Sphingomonadales, Pseudomonadales, Caudobacterales and Rhizobiales). Genomic potential for bioremediation processes, including nitrate removal through aerobic denitrification, and degradation of aromatic compounds and other toxic products were enriched in these microbes. The detrimental side effects were the increased number of antimicrobial resistance genes and the presence of potentially emergent pathogens. Knowledge of this metabolic diversity and the microbes involved in ecological balance recovery can be used to reduce the environmental impact of these practices.

BackgroundThe Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former albeit with lower salinity and (mostly) temperature. In spite of its well-known hydrology and physico chemistry, this enormous water mass remains poorly studied at the microbial genomics level.ResultsWe have sampled its different water masses and analyzed the microbiome by classic and genome-resolved metagenomics generating a large number of metagenome-assembled genomes (MAGs) from them. The oxic zone presents many similarities to the global ocean while the euxinic water mass has similarities to other similar aquatic environments of marine or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environments and includes Cyanobacteria (Synechococcus), photoheterotrophs (largely with marine relatives), facultative/microaerophilic microbes again largely marine, chemolithotrophs (N and S oxidizers) and a large number of anaerobes, mostly sulfate reducers but also a few methanogens and a large number of “dark matter” streamlined genomes of largely unpredictable ecology.ConclusionsThe Black Sea presents a mixture of similarities to other water bodies. The photic zone has many microbes in common with that of the Mediterranean with the relevant exception of the absence of Prochlorococcus. The chemocline already presents very different characteristics with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. Finally the euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy generating metabolism and a small but detectable methanogenesis.We are adding critical information about this unique and important ecosystem and its microbiome.

As the most abundant bacteria in oceans, the Pelagibacterales order (here SAR11) plays an important role in the global carbon cycle, but the study of the evolutionary forces driving its evolution has lagged considerably due to the inherent difficulty of obtaining pure cultures. Multiple evolutionary models have been proposed to explain the diversification of distinct lineages within a population; however, the identification of many of these patterns in natural populations remains mostly enigmatic. We have used a metagenomic approach to explore microdiversity patterns in their natural habitats. Comparison with a collection of bacterial and archaeal groups from the same environments shows that SAR11 populations have a different evolutionary regime, where multiple genotypes coexist within the same population and remain stable over time. Widespread homologous recombination could be one of the main driving factors of this homogenization., The SAR11 clade of Alphaproteobacteria is the most abundant group of planktonic cells in the near-surface epipelagic waters of the ocean, but the mechanisms underlying its exceptional success have not been fully elucidated. Here, we applied a metagenomic approach to explore microdiversity patterns by measuring the accumulation of synonymous and nonsynonymous mutations as well as homologous recombination in populations of SAR11 from different aquatic habitats (marine epipelagic, bathypelagic, and surface freshwater). The patterns of mutation accumulation and recombination were compared to those of other groups of representative marine microbes with multiple ecological strategies that share the same marine habitat, namely, Cyanobacteria (Prochlorococcus and Synechococcus), Archaea (“Candidatus Nitrosopelagicus” and Marine Group II Thalassoarchaea), and some heterotrophic marine bacteria (Alteromonas and Erythrobacter). SAR11 populations showed widespread recombination among distantly related members, preventing divergence leading to a genetically stable population. Moreover, their high intrapopulation sequence diversity with an enrichment in synonymous replacements supports the idea of a very ancient divergence and the coexistence of multiple different clones. However, other microbes analyzed seem to follow different evolutionary dynamics where processes of diversification driven by geographic and ecological instability produce a higher number of nonsynonymous replacements and lower intrapopulation sequence diversity. Together, these data shed light on some of the evolutionary and ecological processes that lead to the large genomic diversity in SAR11. Furthermore, this approach can be applied to other similar microbes that are difficult to culture in the laboratory, but abundant in nature, to investigate the underlying dynamics of their genomic evolution. IMPORTANCE As the most abundant bacteria in oceans, the Pelagibacterales order (here SAR11) plays an important role in the global carbon cycle, but the study of the evolutionary forces driving its evolution has lagged considerably due to the inherent difficulty of obtaining pure cultures. Multiple evolutionary models have been proposed to explain the diversification of distinct lineages within a population; however, the identification of many of these patterns in natural populations remains mostly enigmatic. We have used a metagenomic approach to explore microdiversity patterns in their natural habitats. Comparison with a collection of bacterial and archaeal groups from the same environments shows that SAR11 populations have a different evolutionary regime, where multiple genotypes coexist within the same population and remain stable over time. Widespread homologous recombination could be one of the main driving factors of this homogenization.

Four methicillin-resistant Staphylococcus sciuri (MRSS) strains isolated from stranded dogs showed trimethoprim (TMP) resistance, while all staphylococcal TMP resistant dihydrofolate reductase genes (dfr) were negative. An in-depth whole-genome-sequencing approach on strain C2865 was followed for resistome and mobilome profiling, and for comparative genomics with S. sciuri group available genomes. Lack of species host tropism was observed, with MRSS C2865 placed at a separate sub-branch within S. sciuri species, close to the average nucleotide identity to be considered a different species (95-96%). S. sciuri proved a pronounced accessory genome (73% of genes), while MRSS C2865 distinctively harboured the highest total gene number and highest number of unique genes, with 75% associated to the recognised mobilome. A novel multidrug resistance mosaic plasmid (pUR2865-34) with several adaptive, mobilization (oriT mimic) and segregational stability (Type Ib par system) traits and two small single resistance plasmids were identified. Plasmid pUR2865-34 enclosed a novel staphylococcal TMP resistance gene, named dfrE, which shared highest identity with dfr of soil-related Paenibacillus anaericanus (68%). DfrE conferred high-level TMP resistance in S. aureus and Escherichia coli. Database searches revealed that dfrE was formerly denoted (dfr_like) in an Exiguobacterium spp. from a fish-farm sediment and that was present but unnoticed in several staphylococcal and onemacrococcal genomes with different epidemiological backgrounds. Novel chromosomal site-specific mobile islands with resourceful traits were identified, including a multidrug-resistant SCCmec cassette lacking cassette chromosome recombinase (Ccr) genes, a staphylococcal pathogenicity island of the SaPI4 family, and three unrelated siphoviridae prophages, two of which enclosed recombinases with the conserved Ccr-motif. We reveal a novel staphylococcal TMP resistance dfrE gene already present in diverse bacterial backgrounds. We confirm the ubiquity, high genome plasticity and low host tropism of S. sciuri highlighting its role as a resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.Author summaryStaphylococcus spp. are ubiquitous bacteria present in diverse ecological niches, including humans, animals and the environment. They are clinically relevant opportunistic pathogens and are notorious for their ability to acquire antimicrobial resistance (AMR) and virulence properties, resulting in a significant impact for Public Health. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information across bacterial species. Staphylococcus sciuri represents one of the most ancestral species in the genus and has been suggested a reservoir for AMR genes. Here, following a refined whole genome sequencing approach we determined the entire genome of an animal and environment-associated multidrug resistant (MDR) S. sciuri strain uncovering a novel acquired staphylococcal TMP resistance gene already spread among different bacterial species from different epidemiological backgrounds. We also reveal several additional MGEs, including a novel MDR mobilizable plasmid that encloses several adaptive and stabilization features, and novel mobilizable chromosomal islands with resourceful traits, including three unrelated prophages. Together with comparative genomics, we confirm the ubiquity, high intraspecies heterogenicity, genome plasticity and low host tropism of this species, highlighting its role as resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.

Background Bacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies. Results We here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 106 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity. Conclusion We have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

Marine Euryarchaeota remain among the least understood major components of marine microbial communities. Marine group II Euryarchaeota (MG-II) are more abundant in surface waters (4–20% of the total prokaryotic community), whereas marine group III Euryarchaeota (MG-III) are generally considered low-abundance members of deep mesopelagic and bathypelagic communities. Using genome assembly from direct metagenome reads and metagenomic fosmid clones, we have identified six novel MG-III genome sequence bins from the photic zone (Epi1–6) and two novel bins from deep-sea samples (Bathy1–2). Genome completeness in those genome bins varies from 44% to 85%. Photic-zone MG-III bins corresponded to novel groups with no similarity, and significantly lower GC content, when compared with previously described deep-MG-III genome bins. As found in many other epipelagic microorganisms, photic-zone MG-III bins contained numerous photolyase and rhodopsin genes, as well as genes for peptide and lipid uptake and degradation, suggesting a photoheterotrophic lifestyle. Phylogenetic analysis of these photolyases and rhodopsins as well as their genomic context suggests that these genes are of bacterial origin, supporting the hypothesis of an MG-III ancestor that lived in the dark ocean. Epipelagic MG-III occur sporadically and in relatively small proportions in marine plankton, representing only up to 0.6% of the total microbial community reads in metagenomes. None of the reconstructed epipelagic MG-III genomes were present in metagenomes from aphotic zone depths or from high latitude regions. Most low-GC bins were highly enriched at the deep chlorophyll maximum zones, with the exception of Epi1, which appeared evenly distributed throughout the photic zone worldwide This work was supported by projects MEDIMAX BFPU2013–48007-P from the Spanish Ministerio de Economía y Competitividad MaCuMBA Project 311975 of the European Commission FP7 Project AQUAMET II/2014/012 from the Generalitat Valenciana and by the French Agence Nationale de la Recherche (ANR-08-GENM-024–001,EVOLDEEP). JHM was supported with a PhD fellowship from the Spanish Ministerio de Economía y Competitividad.

We performed an incubation experiment of seawater confined in plastic bottles with samples collected at three depths (15, 60, and 90 m) after retrieval from a single offshore location in the Mediterranean Sea, from a late summer stratified water column. Two samples representative of each depth were collected and stored in opaque bottles after two periods of 7 h. We took advantage of the “bottle effect” to investigate changes in the natural microbial communities (abundant and rare). We recovered 94 metagenome-assembled genomes (MAGs) and 1089 metagenomic viral contigs and examined their abundance using metagenomic recruitment. We detected a significant fast growth of copiotrophic bacteria such as Alteromonas or Erythrobacter throughout the entire water column with different dynamics that we assign to “clonal,” “polyclonal,” or “multispecies” depending on the recruitment pattern. Results also showed a marked ecotype succession in the phototropic picocyanobacteria that were able to grow at all the depths in the absence of light, highlighting the importance of their mixotrophic potential. In addition, “wall-chain-reaction” hypothesis based on the study of phage–host dynamics showed the higher impact of viral predation on archaea in deeper waters, evidencing their prominent role during incubations. Our results provide a step forward in understanding the mechanisms underlying dynamic patterns and ecology of the marine microbiome and the importance of processing the samples immediately after collection to avoid changes in the community structure.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes.

BackgroundBacteriophages are the most numerous biological entities on earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil samples will shed light not only on the yet largely unknown phage diversity, but also may result in novel insights into phage biology and functioning. Unfortunately, the study of soil viromes lags far behind any other ecological model system, due to the heterogeneous soil matrix that rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is therefore a fundamental prerequisite for replicable results and comparative virome studies.ResultsWe here report the optimization of protocols for extraction of bacteriophage DNA from soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with a viral community consisting of phages from different families (106 PFU/g soil): Listeria phage ΦA511 (Myovirus), Staphylococcus phage Φ2638AΔLCR (Siphovirus), and Escherichia phage ΦT7 (Podovirus). The efficacy of bacteriophage (i) elution, (ii) filtration, (iii) concentration, and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for non-amplification dependent sequencing and low 16S rRNA gene contamination levels (≤ 0.2 ‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65 %. In addition, 468 novel circularized soil phage genomes in size up to 235 kb were obtained from over 29,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity.ConclusionWe have shown a dramatically enhanced extraction of the soil phage community by protocol optimization that has proven robustness in both a culture-depended as well as through metaviromic analysis. Our huge data set of manually curated soil viral contigs roughly doubles the amount of currently available soil virome data, and provide insights into the yet largely undescribed soil viral sequence space.

Members of the SAR11 clade, despite their high abundance, are often poorly represented by metagenome‐assembled genomes. This fact has hampered our knowledge about their ecology and genetic diversity. Here we examined 175 SAR11 genomes, including 47 new single‐amplified genomes. The presence of the first genomes associated with subclade IV suggests that, in the same way as subclade V, they might be outside the proposed Pelagibacterales order. An expanded phylogenomic classification together with patterns of metagenomic recruitment at a global scale have allowed us to define new ecogenomic units of classification (genomospecies), appearing at different, and sometimes restricted, metagenomic data sets. We detected greater microdiversity across the water column at a single location than in samples collected from similar depth across the global ocean, suggesting little influence of biogeography. In addition, pangenome analysis revealed that the flexible genome was essential to shape genomospecies distribution. In one genomospecies preferentially found within the Mediterranean, a set of genes involved in phosphonate utilization was detected. While another, with a more cosmopolitan distribution, was unique in having an aerobic purine degradation pathway. Together, these results provide a glimpse of the enormous genomic diversity within this clade at a finer resolution than the currently defined clades. This work was supported by grant ‘VIREVO’ CGL2016-76273-P [AEI/FEDER, EU] (cofounded with FEDER funds) from the Spanish Ministerio de Economía, Industria y Competitividad to FRV, and grants CGL2013-40564-R and SAF2013-49267-EXP from the Spanish Ministerio de Economía, Industria y Competitividad, grant ACIF/2015/332 from Generalitat Valenciana and grant 5334 from the Betty Moore Foundation to MMG. FRV was also a beneficiary of the 5top100-program of the Ministry for Science and Education of Russia. JHM was supported by a Ph.D. fellowship from the Spanish Ministerio de Economía y Competitividad (BES-2014-067828). MLP was supported by a postdoctoral fellowship from the Spanish Ministerio de Economía, Industria y Competitividad (IJCI-2017-34002).

Vibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections., Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel “chromids,” or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.

Marine Group I (MGI) Thaumarchaeota are some of the most abundant microorganisms in the deep ocean and responsible for much of the ammonia oxidation occurring in this environment. In this work, we present 35 sequences assembled from metagenomic samples of the first uncultivated Caudovirales viruses associated with Thaumarchaeota, which we designated marthavirus. Most of the sequences were obtained from cellular metagenomes confirming that they represent an important tool to study environmental viral communities due to cells retrieved while undergoing viral lysis. Metagenomic recruitment showed that this viral population is formed by very divergent entities with high intrapopulation homogeneity. However, metatranscriptomic analyses revealed the same differential expression profile with the capsid as major transcript, indicative of viruses during the lytic cycle. The cobalamine biosynthesis gene cobS, an auxiliary metabolic gene, was also highly expressed during the infection. These analyses expand our understanding of the global diversity of archaeal viruses Valencian Consellería de Educació, Investigació, Cultura i Esport. Grant Number: APOSTD/2016/051 Spanish Ministerio de Economía y Competitividad. Grant Number: BES‐2014‐067828 Generalitat Valenciana. Grant Number: PROMETEO II/2014/012 Spanish Ministerio de Economía, Industria y Competitividad. Grant Number: CGL2015‐71523‐REDC EDER funds. Grant Numbers: VIREVO’ CGL2016‐76273‐P [AEI/FEDER, EU], MEDIMAX’ BFPU2013‐48007‐P

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. This work has been supported by Spanish Ministry of Economy and Competitiveness (refs. CGL2013-40564-R and SAF2013-49267-EXP), Generalitat Valenciana (refs. ACOM/2015/133 and ACIF/2015/332), and Gordon and Betty Moore Foundation (grant 5334).

Freshwater picocyanobacteria including Synechococcus remain poorly studied at the genomic level, compared to their marine representatives. Here, using a metagenomic assembly approach we discovered two novel Synechococcus sp. genomes from two freshwater reservoirs Tous and Lake Lanier, both sharing 96% average nucleotide identity and displaying high abundance levels in these two lakes located at similar altitudes and temperate latitudes. These new genomes have the smallest estimated size (2.2 Mb) and average intergenic spacer length (20 bp) of any previously sequenced freshwater Synechococcus, which may contribute to their success in oligotrophic freshwater systems. Fluorescent in situ hybridization confirmed that Synechococcus sp. Tous comprises small cells (0.987 _ 0.139 mm length, 0.723 _ 0.119 mm width) that amount to 90% of the picocyanobacteria in Tous. They appear together in a phylogenomic tree with Synechococcus sp. RCC307 strain, the main representative of sub-cluster 5.3 that has itself one of the smallest marine Synechococcus genomes. We detected a type II phycobilisome (PBS) gene cluster in both genomes, which suggests that they belong to a phycoerythrin-rich pink low-light ecotype. The decrease of acidic proteins and the higher content of basic transporters and membrane proteins in the novel Synechococcus genomes, compared to marine representatives, support their freshwater specialization. A sulfate Cys transporter which is absent in marine but has been identified in many freshwater cyanobacteria was also detected in Synechococcus sp. Tous. The RuBisCo subunits from this microbe are phylogenetically close to the freshwater amoeba Paulinella chromatophora symbiont, hinting to a freshwater origin of the carboxysome operon of this protist. The novel genomes enlarge the known diversity of freshwater Synechococcus and improve the overall knowledge of the relationships among members of this genus at large This work was supported by projects “MEDIMAX”- BFPU2013- 48007-P, Acciones de dinamización “REDES DE EXCELENCIA” CONSOLIDER- CGL2015-71523-REDC from the Spanish Ministerio de Economía y Competitividad PROMETEO II/2014/012 “AQUAMET” from Generalitat Valenciana. JHM was supported by a Ph.D. fellowship from the Spanish Ministerio de Economía y Competitividad RG was supported by the Grant Agency of the Czech Republic by the research grant 17-04828S.

Most marine metagenomic studies of the marine photic zone analyze only samples taken at one or two depths. However, when the water column is stratified, physicochemical parameters change dramatically over relatively short depth intervals. We sampled the photic water column every 15m depth at a single point of an off-shore Mediterranean site during a period of strong stratification (early autumn) to evaluate the effects of small depth increases on the microbiome. Using genomic assembly and metagenomic read recruitment, we found major shifts in the community structure over small variations of depth, with most microbes showing a distribution limited to layers approximately 30 meters thick (stenobathic). Only some representatives of the SAR11 clade and the Sphingomonadaceae appeared to be eurybathic, spanning a greater range of depths. These results were confirmed by studying a single gene (rhodopsin) for which we also found narrow depth distributions. Our results highlight the importance of considering vertical distribution as a major element when analyzing the presence of marine clades and species or comparing the microbiome present at different locations.

We have used two metagenomic approaches, direct sequencing of natural samples and sequencing after enrichment, to characterize communities of prokaryotes associated to particles. In the first approximation, different size filters (0.22 and 5 μm) were used to identify prokaryotic microbes of free-living and particle-attached bacterial communities in the Mediterranean water column. A subtractive metagenomic approach was used to characterize the dominant microbial groups in the large size fraction that were not present in the free-living one. They belonged mainly to Actinobacteria, Planctomycetes, Flavobacteria and Proteobacteria. In addition, marine microbial communities enriched by incubation with different kinds of particulate material have been studied by metagenomic assembly. Different particle kinds (diatomaceous earth, sand, chitin and cellulose) were colonized by very different communities of bacteria belonging to Roseobacter, Vibrio, Bacteriovorax, and Lacinutrix that were distant relatives of genomes already described from marine habitats. Besides, using assembly from deep metagenomic sequencing from the particle-specific enrichments we were able to determine a total of 20 groups of contigs (eight of them with >50% completeness) and reconstruct de novo five new genomes of novel species within marine clades (>79% completeness and

Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range) obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly, Spanish Ministerio de Economía y Competitividad; and project AQUAMET PROMETEOII/2014/012 from Generalitat Valenciana. JMHM was supported with a PhD fellowship from the Spanish Ministerio de Economía y Competitividad (BES-2014-067828).