Rhodopsin-mediated nutrient uptake by cultivated photoheterotrophic Verrucomicrobiota.

Rhodopsin photosystems convert light energy into electrochemical gradients used by the cell to produce ATP, or for other energy-demanding processes. While these photosystems are widespread in the ocean and have been identified in diverse microbial taxonomic groups, their physiological role in vivo has only been studied in few marine bacterial strains. Recent metagenomic studies revealed the presence of rhodopsin genes in the understudied Verrucomicrobiota phylum, yet their distribution within different Verrucomicrobiota lineages, their diversity, and function remain unknown. In this study, we show that more than 7% of Verrucomicrobiota genomes (n = 2916) harbor rhodopsins of different types. Furthermore, we describe the first two cultivated rhodopsin-containing strains, one harboring a proteorhodopsin gene and the other a xanthorhodopsin gene, allowing us to characterize their physiology under laboratory-controlled conditions. The strains were isolated in a previous study from the Eastern Mediterranean Sea and read mapping of 16S rRNA gene amplicons showed the highest abundances of these strains at the deep chlorophyll maximum (source of their inoculum) in winter and spring, with a substantial decrease in summer. Genomic analysis of the isolates suggests that motility and degradation of organic material, both energy demanding functions, may be supported by rhodopsin phototrophy in Verrucomicrobiota. Under culture conditions, we show that rhodopsin phototrophy occurs under carbon starvation, with light-mediated energy generation supporting sugar transport into the cells. Overall, this study suggests that photoheterotrophic Verrucomicrobiota may occupy an ecological niche where energy harvested from light enables bacterial motility toward organic matter and supports nutrient uptake.

Bacterial aerobic methane cycling by the marine sponge-associated microbiome.

BACKGROUND: Methanotrophy by the sponge-hosted microbiome has been mainly reported in the ecological context of deep-sea hydrocarbon seep niches where methane is either produced geothermically or via anaerobic methanogenic archaea inhabiting the sulfate-depleted sediments. However, methane-oxidizing bacteria from the candidate phylum Binatota have recently been described and shown to be present in oxic shallow-water marine sponges, where sources of methane remain undescribed. RESULTS: Here, using an integrative -omics approach, we provide evidence for sponge-hosted bacterial methane synthesis occurring in fully oxygenated shallow-water habitats. Specifically, we suggest methane generation occurs via at least two independent pathways involving methylamine and methylphosphonate transformations that, concomitantly to aerobic methane production, generate bioavailable nitrogen and phosphate, respectively. Methylphosphonate may be sourced from seawater continuously filtered by the sponge host. Methylamines may also be externally sourced or, alternatively, generated by a multi-step metabolic process where carnitine, derived from sponge cell debris, is transformed to methylamine by different sponge-hosted microbial lineages. Finally, methanotrophs specialized in pigment production, affiliated to the phylum Binatota, may provide a photoprotective function, closing a previously undescribed C1-metabolic loop that involves both the sponge host and specific members of the associated microbial community. CONCLUSION: Given the global distribution of this ancient animal lineage and their remarkable water filtration activity, sponge-hosted methane cycling may affect methane supersaturation in oxic coastal environments. Depending on the net balance between methane production and consumption, sponges may serve as marine sources or sinks of this potent greenhouse gas. Video Abstract.

Functional characterization and taxonomic classification of novel gammaproteobacterial diversity in sponges.

Sponges harbour exceptionally diverse microbial communities, whose members are largely uncultured. The class Gammaproteobacteria often dominates the microbial communities of various sponge species, but most of its diversity remains functional and taxonomically uncharacterised. Here we reconstructed and characterised 32 metagenome-assembled genomes (MAGs) derived from three sponge species. These MAGs represent ten novel species and belong to seven orders, of which one is new. We propose nomenclature for all these taxa. These new species comprise sponge-specific bacteria with varying levels of host specificity. Functional gene profiling highlights significant differences in metabolic capabilities across the ten species, though each also often exhibited a large degree of metabolic diversity involving various nitrogen- and sulfur-based compounds. The genomic features of the ten species suggest they have evolved to form symbiotic interaction with their hosts or are well-adapted to survive within the sponge environment. These Gammaproteobacteria are proposed to scavenge substrates from the host environment, including metabolites or cellular components of the sponge. Their diverse metabolic capabilities may allow for efficient cycling of organic matter in the sponge environment, potentially to the benefit of the host and other symbionts.

Spatiotemporal Variation of Microbial Communities in the Ultra-Oligotrophic Eastern Mediterranean Sea.

Marine microbial communities vary seasonally and spatially, but these two factors are rarely addressed together. In this study, the temporal and spatial patterns of the bacterial and archaeal community were studied along a coast-to-offshore transect in the Eastern Mediterranean Sea (EMS) over six cruises, in three seasons of 2 consecutive years. Amplicon sequencing of 16S rRNA genes and transcripts was performed to determine presence and activity, respectively. The ultra-oligotrophic status of the Southeastern Mediterranean Sea was reflected in the microbial community composition dominated by oligotrophic bacterial groups such as SAR11, even at the most coastal station sampled, throughout the year. Seasons significantly affected the microbial communities, explaining more than half of the observed variability. However, the same few taxa dominated the community over the 2-year sampling period, varying only in their degree of dominance. While there was no overall effect of station location on the microbial community, the most coastal site (16 km offshore) differed significantly in community structure and activity from the three further offshore stations in early winter and summer. Our data on the microbial community compositions and their seasonality support previous notions that the EMS behaves like an oceanic gyre.

Contribution of Maternal and Paternal Transmission to Bacterial Colonization in Nematostella vectensis.

Microbial communities confer multiple beneficial effects to their multicellular hosts. To evaluate the evolutionary and ecological implications of the animal-microbe interactions, it is essential to understand how bacterial colonization is secured and maintained during the transition from one generation to the next. However, the mechanisms of symbiont transmission are poorly studied for many species, especially in marine environments, where the surrounding water constitutes an additional source of microbes. Nematostella vectensis, an estuarine cnidarian, has recently emerged as model organism for studies on host-microbes interactions. Here, we use this model organism to study the transmission of bacterial colonizers, evaluating the contribution of parental and environmental transmission to the establishment of bacterial communities of the offspring. We induced spawning in adult male and female polyps of N. vectensis and used their gametes for five individual fertilization experiments. While embryos developed into primary polyps, we sampled each developmental stage and its corresponding medium samples. By analyzing the microbial community compositions of all samples through 16S rRNA gene amplicon sequencing, we showed that all host tissues harbor microbiota significantly different from the surrounding medium. Interestingly, oocytes and sperms are associated with distinct bacterial communities, indicating the specific vertical transmission of bacterial colonizers by the gametes. These differences were consistent among all the five families analyzed. By overlapping the identified bacterial ASVs associated with gametes, offspring and parents, we identified specific bacterial ASVs that are well supported candidates for vertical transmission via mothers and fathers. This is the first study investigating bacteria transmission in N. vectensis, and among few on marine spawners that do not brood larvae. Our results shed light on the consistent yet distinct maternal and paternal transfer of bacterial symbionts along the different life stages and generations of an aquatic invertebrate.

Particle-associated and free-living bacterial communities in an oligotrophic sea are affected by different environmental factors.

In the oceans and seas, environmental conditions change over multiple temporal and spatial scales. Here, we ask what factors affect the bacterial community structure across time, depth and size fraction during six seasonal cruises (2 years) in the ultra-oligotrophic Eastern Mediterranean Sea. The bacterial community varied most between size fractions (free-living (FL) vs. particle-associated), followed by depth and finally season. The FL community was taxonomically richer and more stable than the particle-associated (PA) one, which was characterized by recurrent 'blooms' of heterotrophic bacteria such as Alteromonas and Ralstonia. The heterotrophic FL and PA communities were also correlated with different environmental parameters: the FL population correlated with depth and phytoplankton, whereas PA bacteria were correlated primarily with the time of sampling. A significant part of the variability in community structure could, however, not be explained by the measured parameters. The metabolic potential of the PA community, predicted from 16S rRNA amplicon data using PICRUSt, was enriched in pathways associated with the degradation and utilization of biological macromolecules, as well as plastics, other petroleum products and herbicides. The FL community was enriched in predicted pathways for the metabolism of inositol phosphate, a potential phosphorus source, and of polycyclic aromatic hydrocarbons.

Microbial rhodopsins are increasingly favoured over chlorophyll in High Nutrient Low Chlorophyll waters.

Microbial rhodopsins are simple light-harvesting complexes that, unlike chlorophyll photosystems, have no iron requirements for their synthesis and phototrophic functions. Here, we report the environmental concentrations of rhodopsin along the Subtropical Frontal Zone off New Zealand, where Subtropical waters encounter the iron-limited Subantarctic High Nutrient Low Chlorophyll (HNLC) region. Rhodopsin concentrations were highest in HNLC waters where chlorophyll-a concentrations were lowest. Furthermore, while the ratio of rhodopsin to chlorophyll-a photosystems was on average 20 along the transect, this ratio increased to over 60 in HNLC waters. We further show that microbial rhodopsins are abundant in both picoplankton (0.2-3 µm) and in the larger (>3 µm) size fractions of the microbial community containing eukaryotic plankton and/or particle-attached prokaryotes. These findings suggest that rhodopsin phototrophy could be critical for microbial plankton to adapt to resource-limiting environments where photosynthesis and possibly cellular respiration are impaired.

Draft Genome Sequence of Terrestrial Streptomyces sp. Strain VITNK9, Isolated from Vellore, Tamil Nadu, India, Exhibiting Antagonistic Activity against Fish Pathogens.

We report the draft genome sequence of Streptomyces sp. strain VITNK9, isolated from a soil sample collected in Vellore District (12.9165°N, 79.1325°E), Tamil Nadu, India, with an assembly size of 7,920,076 bp and 72.7% GC content.

Characterization of sponge-associated Verrucomicrobia: microcompartment-based sugar utilization and enhanced toxin-antitoxin modules as features of host-associated Opitutales.

Bacteria of the phylum Verrucomicrobia are ubiquitous in marine environments and can be found as free-living organisms or as symbionts of eukaryotic hosts. Little is known about host-associated Verrucomicrobia in the marine environment. Here we reconstructed two genomes of symbiotic Verrucomicrobia from bacterial metagenomes derived from the Atlanto-Mediterranean sponge Petrosia ficiformis and three genomes from strains that we isolated from offshore seawater of the Eastern Mediterranean Sea. Phylogenomic analysis of these five strains indicated that they are all members of Verrucomicrobia subdivision 4, order Opitutales. We compared these novel sponge-associated and seawater-isolated genomes to closely related Verrucomicrobia. Genomic analysis revealed that Planctomycetes-Verrucomicrobia microcompartment gene clusters are enriched in the genomes of symbiotic Opitutales including sponge symbionts but not in free-living ones. We hypothesize that in sponge symbionts these microcompartments are used for degradation of l-fucose and l-rhamnose, which are components of algal and bacterial cell walls and therefore may be found at high concentrations in the sponge tissue. Furthermore, we observed an enrichment of toxin-antitoxin modules in symbiotic Opitutales. We suggest that, in sponges, verrucomicrobial symbionts utilize these modules as a defence mechanism against antimicrobial activity deriving from the abundant microbial community co-inhabiting the host.

Sponge microbiome stability during environmental acquisition of highly specific photosymbionts.

In this study, we used in situ transplantations to provide the first evidence of horizontal acquisition of cyanobacterial symbionts by a marine sponge. The acquisition of the symbionts by the host sponge Petrosia ficiformis, which was observed in distinct visible patches, appeared several months after transplantation and at different times on different sponge specimens. We further used 16S rRNA gene amplicon sequencing of genomic DNA (gDNA) and complementary DNA (cDNA) and metatranscriptomics to investigate how the acquisition of the symbiotic cyanobacterium Candidatus Synechococcus feldmannii perturbed the diverse microbiota associated with the host P. ficiformis. To our surprise, the microbiota remained relatively stable during cyanobacterial symbiont acquisition at both structural (gDNA content) and activity (cDNA expression) levels. At the transcriptomic level, photosynthesis was the primary function gained following the acquisition of cyanobacteria. Genes involved in carotene production and oxidative stress tolerance were among those highly expressed by Ca. S. feldmannii, suggesting that this symbiont may protect itself and its host from damaging light radiation.

Identification of Quorum Sensing Activators and Inhibitors in The Marine Sponge Sarcotragus spinosulus.

Marine sponges, a well-documented prolific source of natural products, harbor highly diverse microbial communities. Their extracts were previously shown to contain quorum sensing (QS) signal molecules of the N-acyl homoserine lactone (AHL) type, known to orchestrate bacterial gene regulation. Some bacteria and eukaryotic organisms are known to produce molecules that can interfere with QS signaling, thus affecting microbial genetic regulation and function. In the present study, we established the production of both QS signal molecules as well as QS inhibitory (QSI) molecules in the sponge species Sarcotragus spinosulus. A total of eighteen saturated acyl chain AHLs were identified along with six unsaturated acyl chain AHLs. Bioassay-guided purification led to the isolation of two brominated metabolites with QSI activity. The structures of these compounds were elucidated by comparative spectral analysis of 1HNMR and HR-MS data and were identified as 3-bromo-4-methoxyphenethylamine (1) and 5,6-dibromo-N,N-dimethyltryptamine (2). The QSI activity of compounds 1 and 2 was evaluated using reporter gene assays for long- and short-chain AHL signals (Escherichia coli pSB1075 and E. coli pSB401, respectively). QSI activity was further confirmed by measuring dose-dependent inhibition of proteolytic activity and pyocyanin production in Pseudomonas aeruginosa PAO1. The obtained results show the coexistence of QS and QSI in S. spinosulus, a complex signal network that may mediate the orchestrated function of the microbiome within the sponge holobiont.

Isolation, Genomic and Metabolomic Characterization of Streptomyces tendae VITAKN with Quorum Sensing Inhibitory Activity from Southern India.

Streptomyces are among the most promising genera in terms of production ability to biosynthesize a variety of bioactive secondary metabolites with pharmaceutical interest. Coinciding with the increase in genomic sequencing of these bacteria, mining of their genomes for biosynthetic gene clusters (BGCs) has become a routine component of natural product discovery. Herein, we describe the isolation and characterization of a Streptomyces tendae VITAKN with quorum sensing inhibitory (QSI) activity that was isolated from southern coastal part of India. The nearly complete genome consists of 8,621,231bp with a GC content of 72.2%. Sequence similarity networks of the BGCs detected from this strain against the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) database and 3365 BGCs predicted by antiSMASH analysis of publicly available complete Streptomyces genomes were generated through the BiG-SCAPE-CORASON platform to evaluate its biosynthetic novelty. Crude extract analysis using high-performance liquid chromatography connected to high resolution tandem mass spectrometry (HPLC-HRMS/MS) and dereplication through the Global Natural Product Social Molecular Networking (GNPS) online workflow resulted in the identification of cyclic dipeptides (2, 5-diketopiperazines, DKPs) in the extract, which are known to possess QSI activity. Our results highlight the potential of genome mining coupled with LC-HRMS/MS and in silico tools (GNPS) as a valid approach for the discovery of novel QSI lead compounds. This study also provides the biosynthetic diversity of BGCs and an assessment of the predicted chemical space yet to be discovered.

Genomic Insights Into the Lifestyles of Thaumarchaeota Inside Sponges.

Sponges are among the oldest metazoans and their success is partly due to their abundant and diverse microbial symbionts. They are one of the few animals that have Thaumarchaeota symbionts. Here we compare genomes of 11 Thaumarchaeota sponge symbionts, including three new genomes, to free-living ones. Like their free-living counterparts, sponge-associated Thaumarchaeota can oxidize ammonia, fix carbon, and produce several vitamins. Adaptions to life inside the sponge host include enrichment in transposases, toxin-antitoxin systems and restriction modifications systems, enrichments previously reported also from bacterial sponge symbionts. Most thaumarchaeal sponge symbionts lost the ability to synthesize rhamnose, which likely alters their cell surface and allows them to evade digestion by the host. All but one archaeal sponge symbiont encoded a high-affinity, branched-chain amino acid transporter system that was absent from the analyzed free-living thaumarchaeota suggesting a mixotrophic lifestyle for the sponge symbionts. Most of the other unique features found in sponge-associated Thaumarchaeota, were limited to only a few specific symbionts. These features included the presence of exopolyphosphatases and a glycine cleavage system found in the novel genomes. Thaumarchaeota have thus likely highly specific interactions with their sponge host, which is supported by the limited number of host sponge species to which each of these symbionts is restricted.

Life at Home and on the Roam: Genomic Adaptions Reflect the Dual Lifestyle of an Intracellular, Facultative Symbiont.

"Candidatus Synechococcus feldmannii" is a facultative intracellular symbiont of the Atlanto-Mediterranean sponge Petrosia ficiformis. Genomic information of sponge-associated cyanobacteria derives thus far from the obligate and extracellular symbiont "Candidatus Synechococcus spongiarum." Here we utilized a differential methylation-based approach for bacterial DNA enrichment combined with metagenomics to obtain the first draft genomes of "Ca. Synechococcus feldmannii." By comparative genomics, we revealed that some genomic features (e.g., iron transport mediated by siderophores, eukaryotic-like proteins, and defense mechanisms, like CRISPR-Cas [clustered regularly interspaced short palindromic repeats-associated proteins]) are unique to both symbiont types and absent or rare in the genomes of taxonomically related free-living cyanobacteria. These genomic features likely enable life under the conditions found inside the sponge host. Interestingly, there are many genomic features that are shared by "Ca. Synechococcus feldmannii" and free-living cyanobacteria, while they are absent in the obligate symbiont "Ca. Synechococcus spongiarum." These include genes related to cell surface structures, genetic regulation, and responses to environmental stress, as well as the composition of photosynthetic genes and DNA metabolism. We speculate that the presence of these genes confers on "Ca. Synechococcus feldmannii" its facultative nature (i.e., the ability to respond to a less stable environment when free-living). Our comparative analysis revealed that distinct genomic features depend on the nature of the symbiotic interaction: facultative and intracellular versus obligate and extracellular. IMPORTANCE Given the evolutionary position of sponges as one of the earliest phyla to depart from the metazoan stem lineage, studies on their distinct and exceptionally diverse microbial communities should yield a better understanding of the origin of animal-bacterium interactions. While genomes of several extracellular sponge symbionts have been published, the intracellular symbionts have, so far, been elusive. Here we compare the genomes of two unicellular cyanobacterial sponge symbionts that share an ancestor but followed different evolutionary paths-one became intracellular and the other extracellular. Counterintuitively, the intracellular cyanobacteria are facultative, while the extracellular ones are obligate. By sequencing the genomes of the intracellular cyanobacteria and comparing them to the genomes of the extracellular symbionts and related free-living cyanobacteria, we show how three different cyanobacterial lifestyles are reflected by adaptive genomic features.

Identification and chemical characterization of N-acyl-homoserine lactone quorum sensing signals across sponge species and time.

Marine sponges form symbiotic relationships with complex microbial communities, yet little is known about the mechanisms by which these microbes regulate their behavior through gene expression. Many bacterial communities regulate gene expression using chemical signaling termed quorum sensing. While a few previous studies have shown presence of N-acyl-homoserine lactone (AHL)-based quorum sensing in marine sponges, the chemical identity of AHL signals has been published for only two sponge species. In this study, we screened for AHLs in extracts from 15 sponge species (109 specimens in total) from the Mediterranean and Red Sea, using a wide-range AHL biosensor. This is the first time that AHL presence was examined over time in sponges. We detected the presence of AHL in 46% of the sponge species and found that AHL signals differ for certain sponge species in time and across sponge individuals. Furthermore, for the Mediterranean sponge species Sarcotragus fasciculatus, we identified 14 different AHLs. The constant presence of specific AHL molecules in all specimens, together with varying signaling molecules between the different specimens, makes Sa. fasciculatus a good model to further investigate the function of quorum sensing in sponge-associated bacteria. This study extends the knowledge of AHL-based quorum sensing in marine sponges.

Author Correction: Surface properties of SAR11 bacteria facilitate grazing avoidance.

In the version of this Letter originally published, the authors incorrectly stated that primers 28F-519R were reported in ref. 54 to underestimate the abundance of SAR11 in the ocean. This statement has now been amended in all versions of the Letter.

Surface properties of SAR11 bacteria facilitate grazing avoidance.

Oceanic ecosystems are dominated by minute microorganisms that play a major role in food webs and biogeochemical cycles 1 . Many microorganisms thrive in the dilute environment due to their capacity to locate, attach to, and use patches of nutrients and organic matter 2,3 . We propose that some free-living planktonic bacteria have traded their ability to stick to nutrient-rich organic particles for a non-stick cell surface that helps them evade predation by mucous filter feeders. We used a combination of in situ sampling techniques and next-generation sequencing to study the biological filtration of microorganisms at the phylotype level. Our data indicate that some marine bacteria, most notably the highly abundant Pelagibacter ubique and most other members of the SAR 11 clade of the Alphaproteobacteria, can evade filtration by slipping through the mucous nets of both pelagic and benthic tunicates. While 0.3 µm polystyrene beads and other similarly-sized bacteria were efficiently filtered, SAR11 members were not captured. Reversed-phase chromatography revealed that most SAR11 bacteria have a much less hydrophobic cell surface than that of other planktonic bacteria. Our data call for a reconsideration of the role of surface properties in biological filtration and predator-prey interactions in aquatic systems.

The sponge microbiome project.

Marine sponges (phylum Porifera) are a diverse, phylogenetically deep-branching clade known for forming intimate partnerships with complex communities of microorganisms. To date, 16S rRNA gene sequencing studies have largely utilised different extraction and amplification methodologies to target the microbial communities of a limited number of sponge species, severely limiting comparative analyses of sponge microbial diversity and structure. Here, we provide an extensive and standardised dataset that will facilitate sponge microbiome comparisons across large spatial, temporal, and environmental scales. Samples from marine sponges (n = 3569 specimens), seawater (n = 370), marine sediments (n = 65) and other environments (n = 29) were collected from different locations across the globe. This dataset incorporates at least 268 different sponge species, including several yet unidentified taxa. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA using standardised procedures. Raw sequences (total of 1.1 billion sequences) were processed and clustered with (i) a standard protocol using QIIME closed-reference picking resulting in 39 543 operational taxonomic units (OTU) at 97% sequence identity, (ii) a de novo clustering using Mothur resulting in 518 246 OTUs, and (iii) a new high-resolution Deblur protocol resulting in 83 908 unique bacterial sequences. Abundance tables, representative sequences, taxonomic classifications, and metadata are provided. This dataset represents a comprehensive resource of sponge-associated microbial communities based on 16S rRNA gene sequences that can be used to address overarching hypotheses regarding host-associated prokaryotes, including host specificity, convergent evolution, environmental drivers of microbiome structure, and the sponge-associated rare biosphere.