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ABSTRACT Prokaryotic species are composed of multiple clonal lineages coexisting in the same habitat. In recent years, understanding the maintenance of this high intraspecific genetic diversity in asexual microorganisms has been a challenge for microbial ecology. In this study, we investigated the potential ecological role of this genomic heterogeneity in the marine HIMB59 clade. The metagenomic recruitment revealed the presence of three main genomospecies with different ecological distribution patterns within the two defined families. Genomic analysis revealed the presence of a flexible genomic island conserved throughout the order at the same position in the genome and related to phosphate (P) metabolism. The different versions of this island showed a distribution correlated with the concentration of P in the environment but not with the phylogeny at the genomospecies level. At high P availability (>0.5 µM), HIMB59 cells had only the high-affinity phosphate transporter operon (PstSCAB and PhoU). Under conditions of higher P scarcity (

Abstract Background Lake Baikal, the world’s deepest freshwater lake, contains important numbers of Candidatus Patescibacteria (formerly CPR) in its deepest reaches. However, previously obtained CPR metagenome-assembled genomes recruited very poorly indicating the potential of other groups being present. Here, we have applied for the first time a long-read (PacBio CCS) metagenomic approach to analyze in depth the Ca. Patescibacteria living in the bathypelagic water column of Lake Baikal at 1600 m. Results The retrieval of nearly complete 16S rRNA genes before assembly has allowed us to detect the presence of a novel and a likely endemic group of Ca. Patescibacteria inhabiting bathypelagic Lake Baikal. This novel group seems to possess extremely high intra-clade diversity, precluding complete genomes' assembly. However, read binning and scaffolding indicate that these microbes are similar to other Ca. Patescibacteria (i.e. parasites or symbionts), although they seem to carry more anabolic pathways, likely reflecting the extremely oligotrophic habitat they inhabit. The novel bins have not been found anywhere, but one of the groups appears in small amounts in an oligotrophic and deep alpine Lake Thun. We propose this novel group be named Baikalibacteria. Conclusion The recovery of 16S rRNA genes via long-read metagenomics plus the use of long-read binning to uncover highly diverse “hidden” groups of prokaryotes are key strategies to move forward in ecogenomic microbiology. The novel group possesses enormous intraclade diversity akin to what happens with Ca. Patescibacteria at the interclade level, which is remarkable in an environment that has changed little in the last 25 million years.

Summary: Marine group II (MGII) is the most abundant planktonic heterotrophic archaea in the ocean. The evolutionary history of MGII archaea is elusive. In this study, 13 new MGII metagenome-assembled genomes were recovered from surface to the hadal zone in Challenger Deep of the Mariana Trench; four of them from the deep ocean represent a novel group. The optimal growth temperature (OGT) of the common ancestor of MGII has been estimated to be at about 60°C and OGTs of MGIIc, MGIIb, and MGIIa at 47°C–50ºC, 37°C–44ºC, and 30°C–37ºC, respectively, suggesting the adaptation of these species to different temperatures during evolution. The estimated OGT range of MGIIc was supported by experimental measurements of cloned β-galactosidase that showed optimal enzyme activity around 50°C. These results indicate that MGIIc may have originated from a common ancestor that lived in warm or even hot marine environment, such as hydrothermal vents.

ABSTRACT Microbial rhodopsins are found more than once in a single genome (paralogs) often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59, and the Gammaproteobacteria Pseudothioglobus SAGs. These genomes always had a bona fide proteorhodopsin and a separate cluster of genes containing a second rhodopsin associated with a predicted flotillin coding gene and have thus been named flotillin-associated rhodopsins (FArhodopsins). Although they are members of the proteorhodopsin protein family, they form a separate clade within that family and are quite divergent from known proton-pumping proteorhodopsins. They contain either DTT, DTL, or DNI motifs in their key functional amino acids. FArhodopsins are mainly associated with the lower layers of the epipelagic zone. All marine FArhodopsins had the retinal binding lysine, but we found relatives in freshwater metagenomes lacking this key amino acid. AlphaFold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less. Freshwater FArhodopsins were more diverse than marine ones, but we could not determine if there were other rhodopsins in the genome due to the lack of SAGs or isolates. Although the function of FArhodopsins could not be established, their conserved genomic context indicated involvement in the formation of membrane microdomains. The conservation of FArhodopsins in diverse and globally abundant microorganisms suggests that they may be important in the adaptation to the twilight zone of aquatic environments. IMPORTANCE Rhodopsins have been shown to play a key role in the ecology of aquatic microbes. Here, we describe a group of widespread rhodopsins in aquatic microbes associated with dim light conditions. Their characteristic genomic context found in both marine and freshwater environments indicates a novel potential involvement in membrane microstructure that could be important for the function of the coexisting proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to a drastically different physiological role.

The deep ocean, one of the largest ecosystems on earth, is dominated by microorganisms that are keystones in the regulation of biogeochemical cycles. However, the evolutionary pathways underlying the specific adaptations required (e.g., high pressure and low temperature) by this unique niche remain understudied. Here, we analyzed the first representatives belonging to the order Acidimicrobiales, a group of marine planktonic Actinobacteriota, that specifically inhabits the aphotic zone of the oceanic water column (>200 m). Compared with their epipelagic counterparts, deep-sea representatives showed the same evolution in genome architecture with higher GC content, longer intergenic spaces as well as higher nitrogen (N-ARSC) and lower carbon (C-ARSC) content in encoded amino acid residue side chains consistent with the higher nitrogen concentration and lower carbon concentration in deep waters compared to the photic zone. Metagenomic recruitment showed distribution patterns that allowed the description of different ecogenomic units within the three deep water-associated genera defined by our phylogenomic analyses (UBA3125, S20-B6 and UBA9410). The entire genus UBA3125 was found exclusively associated with oxygen minimum zones linked to the acquisition of genes involved in denitrification. Genomospecies of genus S20-B6 recruited in samples from both mesopelagic (200–1,000 m) and bathypelagic (1000–4,000 m) zones, including polar regions. Diversity in the genus UBA9410 was higher, with genomospecies widely distributed in temperate zones, others in polar regions, and the only genomospecies associated with abyssal zones (>4,000 m). At the functional level, groups beyond the epipelagic zone have a more complex transcriptional regulation including in their genomes a unique WhiB paralog. In addition, they showed higher metabolic potential for organic carbon and carbohydrate degradation as well as the ability to accumulate glycogen as a source of carbon and energy. This could compensate for energy metabolism in the absence of rhodopsins, which is only present in genomes associated with the photic zone. The abundance in deep samples of cytochrome P450 monooxygenases associated with the genomes of this order suggests an important role in remineralization of recalcitrant compounds throughout the water column.

Background: Pseudomonas aeruginosa shows resistance to several antibiotics and often develops such resistance during patient treatment. Objective: Develop an in vitro model, using clinical isolates of P. aeruginosa, to compare the ability of the imipenem and imipenem/relebactam to generate resistant mutants to imipenem and to other antibiotics. Perform a genotypic analysis to detect how the selective pressure changes their genomes. Methods: The antibiotics resistance was studied by microdilution assays and e-test, and the genotypic study was performed by NGS. Results: The isolates acquired resistance to imipenem in an average of 6 days, and to imipenem/relebactam in 12 days (p value = 0.004). After 30 days of exposure, 75% of the isolates reached a MIC > 64 mg/L for imipenem and 37.5% for imipenem/relebactam (p value = 0.077). The 37.5% and the 12.5% imipenem/relebactam mutants developed resistance to piperacillin/tazobactam and ceftazidime, respectively, while the 87.5% and 37.5% of the imipenem mutants showed resistance to these drugs (p value = 0.003, p value = 0.015). The main biological processes altered by the SNPs were the glycosylation pathway, transcriptional regulation, histidine kinase response, porins, and efflux pumps. Discussion: The addition of relebactam delays the generation of resistance to imipenem and limits the cross-resistance to other beta-lactams. The clinical relevance of this phenomenon, which has the limitation that it has been performed in vitro, should be evaluated by stewardship programs in clinical practice, as it could be useful in controlling multi-drug resistance in P. aeruginosa.

Abstract 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.

ABSTRACT 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). IMPORTANCE We explored the vast genetic diversity of environmental viruses by using a combination of cellular metagenome (as opposed to virome) sequencing using high-fidelity long-read sequences (in this case, PacBio CCS). This approach resulted in the recovery of a representative sample of the viral population, and it performed better (more phage contigs, larger average contig size) than Illumina sequencing applied to the same sample. By this approach, the many biases of assembly are avoided, as the CCS reads recovers (typically around 5 kb) complete genes and even operons, resulting in a better discovery of the viral gene diversity based on viral marker proteins. Thus, biotechnologically promising genes, such as endolysin genes, can be very efficiently searched with this approach. In addition, hybrid assembly produces more complete and longer contigs, which is particularly important for studying little-known viral groups such as the nucleocytoplasmic large DNA viruses (NCLDV).

Abstract 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.

ABSTRACT 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.

ABSTRACT 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 (

The SAR116 clade of Alphaproteobacteria

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.

BackgroundSARS-CoV-2 is an RNA virus causing COVID-19. The clinical characteristics and epidemiology of COVID-19 have been extensively investigated, however, only one study so far focused on the patient’s nasopharynx microbiota. In this study we investigated the nasopharynx microbial community of patients that developed different severity levels of COVID-19. We performed 16S ribosomal DNA 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 microbiota diversity and OTU composition among severity groups and built bacterial co-abundance networks for each group.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 Bacteroidota (18) and Firmicutes (25). OTUs classified as Prevotella were found to be significantly more abundant in patients that developed more severe COVID-19. Furthermore, co-abundance analysis indicated a loss of network complexity among samples from patients that later developed more severe symptoms.ConclusionOur study shows that the nasopharyngeal microbiome of COVID-19 patients showed differences in the composition of specific OTUs and complexity of co-abundance networks. Taxa 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.

ABSTRACT “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.

ABSTRACT 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.

Abstract Background The photic zone of aquatic habitats is subjected to strong physicochemical gradients. To analyze the fine-scale variations in the marine microbiome, we collected seven samples from a single offshore location in the Mediterranean at 15 m depth intervals during a period of strong stratification, as well as two more samples during the winter when the photic water column was mixed. We were able to recover 94 new metagenome-assembled genomes (MAGs) from these metagenomes and examine the distribution of key marine microbes within the photic zone using metagenomic recruitment. Results Our results showed significant differences in the microbial composition of different layers within the stratified photic water column. The majority of microorganisms were confined to discreet horizontal layers of no more than 30 m (stenobathic). Only a few such as members of the SAR11 clade appeared at all depths (eurybathic). During the winter mixing period, only some groups of bloomers such as Pseudomonas were favored. Although most microbes appeared in both seasons, some groups like the SAR116 clade and some Bacteroidetes and Verrucomicrobia seemed to disappear during the mixing period. Furthermore, we found that some microbes previously considered seasonal (e.g., Archaea or Actinobacteria) were living in deeper layers within the photic zone during the stratification period. A strong depth-related specialization was detected, not only at the taxonomic level but also at the functional level, even within the different clades, for the manipulation and uptake of specific polysaccharides. Rhodopsin sequences (green or blue) also showed narrow depth distributions that correlated with the taxonomy of the microbe in which they were found but not with depth. Conclusions Although limited to a single location in the Mediterranean, this study has profound implications for our understanding of how marine microbial communities vary with depth within the photic zone when stratified. Our results highlight the importance of collecting samples at different depths in the water column when comparing seasonal variations and have important ramifications for global marine studies that most often take samples from only one single depth. Furthermore, our perspective and approaches (metagenomic assembly and recruitment) are broadly applicable to other metagenomic studies.

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.

ABSTRACT 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. IMPORTANCE 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.

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 μm length, 0.723 ± 0.119 μm 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.

The “One Health” approach provides a comprehensive framework for understanding antimicrobial resistance. This perspective is of particular importance in the study of Pseudomonas aeruginosa, as it is not only a pathogen that affects humans but also persists in environmental reservoirs. To assess evolutionary selection for niche-specific traits, a genomic comparison of 749 P. aeruginosa strains from three environments (clinical, aquatic, and soil) was performed. The results showed that the environment does indeed exert selective pressure on specific traits. The high percentage of persistent genome, the lack of correlation between phylogeny and origin of the isolate, and the high intrinsic resistance indicate that the species has a high potential for pathogenicity and resistance, regardless of the reservoir. The flexible genome showed an enrichment of metal resistance genes, which could act as a co-selection of antibiotic resistance genes. In the plasmids, resistance genes were found in multigenic clusters, with the presence of a mobile integron being prominent. This integron was identified in several pathogenic strains belonging to distantly related taxa with a worldwide distribution, showing the risk of rapid evolution of resistance. These results provide a more complete understanding of the evolution of P. aeruginosa, which could help develop new prevention strategies.

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.