Distantly related Alteromonas bacteriophages share tail fibers exhibiting properties of transient chaperone caps.

The host recognition modules encoding the injection machinery and receptor binding proteins (RBPs) of bacteriophages are predisposed to mutation and recombination to maintain infectivity towards co-evolving bacterial hosts. In this study, we reveal how Alteromonas mediterranea schitovirus A5 shares its host recognition module, including tail fiber and cognate chaperone, with phages from distantly related families including Alteromonas myovirus V22. While the V22 chaperone is essential for producing active tail fibers, here we demonstrate production of functional A5 tail fibers regardless of chaperone co-expression. AlphaFold-generated models of tail fiber and chaperone pairs from phages A5, V22, and other Alteromonas phages reveal how amino acid insertions within both A5-like proteins results in a knob domain duplication in the tail fiber and a chaperone ß-hairpin "tentacle" extension. These structural modifications are linked to differences in chaperone dependency between the A5 and V22 tail fibers. Structural similarity between the chaperones and intramolecular chaperone domains of other phage RBPs suggests an additional function of these chaperones as transient fiber "caps". Finally, our identification of homologous host recognition modules from morphologically distinct phages implies that horizontal gene transfer and recombination events between unrelated phages may be a more common process than previously thought among Caudoviricetes phages.

Evolutionary pathways for deep-sea adaptation in marine planktonic Actinobacteriota.

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.

Single-amplified genomes reveal most streamlined free-living marine bacteria.

Evolutionary adaptations of prokaryotes to the environment sometimes result in genome reduction. Our knowledge of this phenomenon among free-living bacteria remains scarce. We address the dynamics and limits of genome reduction by examining one of the most abundant bacteria in the ocean, the SAR86 clade. Despite its abundance, comparative genomics has been limited by the absence of pure cultures and the poor representation in metagenome-assembled genomes. We co-assembled multiple previously available single-amplified genomes to obtain the first complete genomes from members of the four families. All families showed a convergent evolutionary trajectory with characteristic features of streamlined genomes, most pronounced in the TMED112 family. This family has a genome size of ca. 1 Mb and only 1 bp as median intergenic distance, exceeding values found in other abundant microbes such as SAR11, OM43 and Prochlorococcus. This genomic simplification led to a reduction in the biosynthesis of essential molecules, DNA repair-related genes, and the ability to sense and respond to environmental factors, which could suggest an evolutionary dependence on other co-occurring microbes for survival (Black Queen hypothesis). Therefore, these reconstructed genomes within the SAR86 clade provide new insights into the limits of genome reduction in free-living marine bacteria.

Elucidating the picocyanobacteria salinity divide through ecogenomics of new freshwater isolates.

BACKGROUND: Cyanobacteria are the major prokaryotic primary producers occupying a range of aquatic habitats worldwide that differ in levels of salinity, making them a group of interest to study one of the major unresolved conundrums in aquatic microbiology which is what distinguishes a marine microbe from a freshwater one? We address this question using ecogenomics of a group of picocyanobacteria (cluster 5) that have recently evolved to inhabit geographically disparate salinity niches. Our analysis is made possible by the sequencing of 58 new genomes from freshwater representatives of this group that are presented here, representing a 6-fold increase in the available genomic data. RESULTS: Overall, freshwater strains had larger genomes (≈2.9 Mb) and %GC content (≈64%) compared to brackish (2.69 Mb and 64%) and marine (2.5 Mb and 58.5%) isolates. Genomic novelties/differences across the salinity divide highlighted acidic proteomes and specific salt adaptation pathways in marine isolates (e.g., osmolytes/compatible solutes - glycine betaine/ggp/gpg/gmg clusters and glycerolipids glpK/glpA), while freshwater strains possessed distinct ion/potassium channels, permeases (aquaporin Z), fatty acid desaturases, and more neutral/basic proteomes. Sulfur, nitrogen, phosphorus, carbon (photosynthesis), or stress tolerance metabolism while showing distinct genomic footprints between habitats, e.g., different types of transporters, did not obviously translate into major functionality differences between environments. Brackish microbes show a mixture of marine (salt adaptation pathways) and freshwater features, highlighting their transitional nature. CONCLUSIONS: The plethora of freshwater isolates provided here, in terms of trophic status preference and genetic diversity, exemplifies their ability to colonize ecologically diverse waters across the globe. Moreover, a trend towards larger and more flexible/adaptive genomes in freshwater picocyanobacteria may hint at a wider number of ecological niches in this environment compared to the relatively homogeneous marine system.

α-cyanobacteria possessing form IA RuBisCO globally dominate aquatic habitats.

RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) is one the most abundant enzymes on Earth. Virtually all food webs depend on its activity to supply fixed carbon. In aerobic environments, RuBisCO struggles to distinguish efficiently between CO2 and O2. To compensate, organisms have evolved convergent solutions to concentrate CO2 around the active site. The genetic engineering of such inorganic carbon concentrating mechanisms (CCMs) into plants could help facilitate future global food security for humankind. In bacteria, the carboxysome represents one such CCM component, of which two independent forms exist: α and ß. Cyanobacteria are important players in the planet's carbon cycle and the vast majority of the phylum possess a ß-carboxysome, including most cyanobacteria used as laboratory models. The exceptions are the exclusively marine Prochlorococcus and Synechococcus that numerically dominate open ocean systems. However, the reason why marine systems favor an α-form is currently unknown. Here, we report the genomes of 58 cyanobacteria, closely related to marine Synechococcus that were isolated from freshwater lakes across the globe. We find all these isolates possess α-carboxysomes accompanied by a form 1A RuBisCO. Moreover, we demonstrate α-cyanobacteria dominate freshwater lakes worldwide. Hence, the paradigm of a separation in carboxysome type across the salinity divide does not hold true, and instead the α-form dominates all aquatic systems. We thus question the relevance of ß-cyanobacteria as models for aquatic systems at large and pose a hypothesis for the reason for the success of the α-form in nature.

Phylogenomics of SAR116 Clade Reveals Two Subclades with Different Evolutionary Trajectories and an Important Role in the Ocean Sulfur Cycle.

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 (<22 bp), and strong purifying selection (low ratio of nonsynonymous to synonymous polymorphisms [dN/dS]). Besides, they were more abundant in metagenomic databases recruiting 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 metagenome-assembled genomes (MAGs), included the only two pure cultures. Genomic analysis suggested that both clades have a significant role in the sulfur cycle with differences in the way both clades can metabolize dimethylsulfoniopropionate (DMSP). IMPORTANCE The SAR116 clade of Alphaproteobacteria is a 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 these kinds 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 genomic makeup of the two subclades that could serve to illustrate 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.

The Resistome and Mobilome of Multidrug-Resistant Staphylococcus sciuri C2865 Unveil a Transferable Trimethoprim Resistance Gene, Designated dfrE, Spread Unnoticed.

Methicillin-resistant Staphylococcus sciuri (MRSS) strain C2865 from a stranded dog in Nigeria was trimethoprim (TMP) resistant but lacked formerly described staphylococcal TMP-resistant dihydrofolate reductase genes (dfr). Whole-genome sequencing, comparative genomics, and pan-genome analyses were pursued to unveil the molecular bases for TMP resistance via resistome and mobilome profiling. MRSS C2865 comprised a species subcluster and positioned just above the intraspecies boundary. Lack of species host tropism was observed. S. sciuri exhibited an open pan-genome, while MRSS C2865 harbored the highest number of unique genes (75% associated with mobilome). Within this fraction, we discovered a transferable TMP resistance gene, named dfrE, which confers high-level TMP resistance in Staphylococcus aureus and Escherichia coli. dfrE was located in a novel multidrug resistance mosaic plasmid (pUR2865-34) encompassing adaptive, mobilization, and segregational stability traits. dfrE was formerly denoted as dfr_like in Exiguobacterium spp. from fish farm sediment in China but escaped identification in one macrococcal and diverse staphylococcal genomes in different Asian countries. dfrE shares the highest identity with dfr of soil-related Paenibacillus anaericanus (68%). Data analysis discloses that dfrE has emerged from a single ancestor and places S. sciuri as a plausible donor. C2865 unique fraction additionally enclosed novel chromosomal mobile islands, including a multidrug-resistant pseudo-SCCmec cassette, three apparently functional prophages (Siphoviridae), and an SaPI4-related staphylococcal pathogenicity island. Since dfrE seems not yet common in staphylococcal clinical specimens, our data promote early surveillance and enable molecular diagnosis. We evidence the genome plasticity of S. sciuri and highlight its role as a resourceful reservoir for adaptive traits. IMPORTANCE The discovery and surveillance of antimicrobial resistance genes (AMRG) and their mobilization platforms are critical to understand the evolution of bacterial resistance and to restrain further expansion. Limited genomic data are available on Staphylococcus sciuri; regardless, it is considered a reservoir for critical AMRG and mobile elements. We uncover a transferable staphylococcal TMP resistance gene, named dfrE, in a novel mosaic plasmid harboring additional resistance, adaptive, and self-stabilization features. dfrE is present but evaded detection in diverse species from varied sources geographically distant. Our analyses evidence that the dfrE-carrying element has emerged from a single ancestor and position S. sciuri as the donor species for dfrE spread. We also identify novel mobilizable chromosomal islands encompassing AMRG and three unrelated prophages. We prove high intraspecies heterogenicity and genome plasticity for S. sciuri. This work highlights the importance of genome-wide ecological studies to facilitate identification, characterization, and evolution routes of bacteria adaptive features.

The microbiome of the Black Sea water column analyzed by shotgun and genome centric metagenomics.

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.

Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition.

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas-targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP.IMPORTANCE Host recognition by phage-encoded receptor binding proteins (RBPs) constitutes the first step in all phage infections and the most critical determinant of host specificity. By characterizing new types of RBPs and identifying their essential chaperones, we hope to expand the repertoire of known phage-host recognition machineries. Due to their genetic plasticity, studying RBPs and their associated chaperones can shed new light onto viral evolution affecting phage-host interactions, which is essential for fields such as phage therapy or biotechnology. In addition, since marine phages constitute one of the most important reservoirs of noncharacterized genetic diversity on the planet, their genomic and functional characterization may be of paramount importance for the discovery of novel genes with potential applications.

Ecogenomics of the SAR11 clade.

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.

Ecological and genomic features of two widespread freshwater picocyanobacteria.

We present two genomes of widespread freshwater picocyanobacteria isolated by extinction dilution from a Spanish oligotrophic reservoir. Based on microscopy and genomic properties, both picocyanobacteria were tentatively designated Synechococcus lacustris Tous, formerly described as a metagenome assembled genome (MAG) from the same habitat, and Cyanobium usitatum Tous, described here for the first time. Both strains were purified in unicyanobacterial cultures, and their genomes were sequenced. They are broadly distributed in freshwater systems; the first seems to be a specialist on temperate reservoirs (Tous, Amadorio, Dexter, Lake Lanier, Sparkling), and the second appears to also be abundant in cold environments including ice-covered lakes such as Lake Baikal, Lake Erie or the brackish Baltic Sea. Having complete genomes provided access to the flexible genome that does not assemble in MAGs. We found several genomic islands in both genomes, within which there were genes for nitrogen acquisition, transporters for a wide set of compounds and biosynthesis of phycobilisomes in both strains. Some of these regions of low coverage in metagenomes also included antimicrobial compounds, transposases and phage defence systems, including a novel type III CRISPR-Cas phage defence system that was only detected in Synechococcus lacustris Tous.