Genome-resolved metagenomics of Venice Lagoon surface sediment bacteria reveals high biosynthetic potential and metabolic plasticity as successful strategies in an impacted environment.

Bacteria living in sediments play essential roles in marine ecosystems and deeper insights into the ecology and biogeochemistry of these largely unexplored organisms can be obtained from 'omics' approaches. Here, we characterized metagenome-assembled-genomes (MAGs) from the surface sediment microbes of the Venice Lagoon (northern Adriatic Sea) in distinct sub-basins exposed to various natural and anthropogenic pressures. MAGs were explored for biodiversity, major marine metabolic processes, anthropogenic activity-related functions, adaptations at the microscale, and biosynthetic gene clusters. Starting from 126 MAGs, a non-redundant dataset of 58 was compiled, the majority of which (35) belonged to (Alpha- and Gamma-) Proteobacteria. Within the broad microbial metabolic repertoire (including C, N, and S metabolisms) the potential to live without oxygen emerged as one of the most important features. Mixotrophy was also found as a successful lifestyle. Cluster analysis showed that different MAGs encoded the same metabolic patterns (e.g., C fixation, sulfate oxidation) thus suggesting metabolic redundancy. Antibiotic and toxic compounds resistance genes were coupled, a condition that could promote the spreading of these genetic traits. MAGs showed a high biosynthetic potential related to antimicrobial and biotechnological classes and to organism defense and interactions as well as adaptive strategies for micronutrient uptake and cellular detoxification. Our results highlighted that bacteria living in an impacted environment, such as the surface sediments of the Venice Lagoon, may benefit from metabolic plasticity as well as from the synthesis of a wide array of secondary metabolites, promoting ecosystem resilience and stability toward environmental pressures. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00192-z.

Comparative Population Transcriptomics Provide New Insight into the Evolutionary History and Adaptive Potential of World Ocean Krill.

Genetic variation is instrumental for adaptation to changing environments but it is unclear how it is structured and contributes to adaptation in pelagic species lacking clear barriers to gene flow. Here, we applied comparative genomics to extensive transcriptome datasets from 20 krill species collected across the Atlantic, Indian, Pacific, and Southern Oceans. We compared genetic variation both within and between species to elucidate their evolutionary history and genomic bases of adaptation. We resolved phylogenetic interrelationships and uncovered genomic evidence to elevate the cryptic Euphausia similis var. armata into species. Levels of genetic variation and rates of adaptive protein evolution vary widely. Species endemic to the cold Southern Ocean, such as the Antarctic krill Euphausia superba, showed less genetic variation and lower evolutionary rates than other species. This could suggest a low adaptive potential to rapid climate change. We uncovered hundreds of candidate genes with signatures of adaptive evolution among Antarctic Euphausia but did not observe strong evidence of adaptive convergence with the predominantly Arctic Thysanoessa. We instead identified candidates for cold-adaptation that have also been detected in Antarctic fish, including genes that govern thermal reception such as TrpA1. Our results suggest parallel genetic responses to similar selection pressures across Antarctic taxa and provide new insights into the adaptive potential of important zooplankton already affected by climate change.

The Rhodoexplorer Platform for Red Algal Genomics and Whole-Genome Assemblies for Several Gracilaria Species.

Macroalgal (seaweed) genomic resources are generally lacking as compared with other eukaryotic taxa, and this is particularly true in the red algae (Rhodophyta). Understanding red algal genomes is critical to understanding eukaryotic evolution given that red algal genes are spread across eukaryotic lineages from secondary endosymbiosis and red algae diverged early in the Archaeplastids. The Gracilariales is a highly diverse and widely distributed order including species that can serve as ecosystem engineers in intertidal habitats and several notorious introduced species. The genus Gracilaria is cultivated worldwide, in part for its production of agar and other bioactive compounds with downstream pharmaceutical and industrial applications. This genus is also emerging as a model for algal evolutionary ecology. Here, we report new whole-genome assemblies for two species (Gracilaria chilensis and Gracilaria gracilis), a draft genome assembly of Gracilaria caudata, and genome annotation of the previously published Gracilaria vermiculophylla genome. To facilitate accessibility and comparative analysis, we integrated these data in a newly created web-based portal dedicated to red algal genomics (https://rhodoexplorer.sb-roscoff.fr). These genomes will provide a resource for understanding algal biology and, more broadly, eukaryotic evolution.

Differential global distribution of marine picocyanobacteria gene clusters reveals distinct niche-related adaptive strategies.

The ever-increasing number of available microbial genomes and metagenomes provides new opportunities to investigate the links between niche partitioning and genome evolution in the ocean, especially for the abundant and ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus. Here, by combining metagenome analyses of the Tara Oceans dataset with comparative genomics, including phyletic patterns and genomic context of individual genes from 256 reference genomes, we show that picocyanobacterial communities thriving in different niches possess distinct gene repertoires. We also identify clusters of adjacent genes that display specific distribution patterns in the field (eCAGs) and are thus potentially involved in the same metabolic pathway and may have a key role in niche adaptation. Several eCAGs are likely involved in the uptake or incorporation of complex organic forms of nutrients, such as guanidine, cyanate, cyanide, pyrimidine, or phosphonates, which might be either directly used by cells, for example for the biosynthesis of proteins or DNA, or degraded to inorganic nitrogen and/or phosphorus forms. We also highlight the enrichment of eCAGs involved in polysaccharide capsule biosynthesis in Synechococcus populations thriving in both nitrogen- and phosphorus-depleted areas vs. low-iron (Fe) regions, suggesting that the complexes they encode may be too energy-consuming for picocyanobacteria thriving in the latter areas. In contrast, Prochlorococcus populations thriving in Fe-depleted areas specifically possess an alternative respiratory terminal oxidase, potentially involved in the reduction of Fe(III) to Fe(II). Altogether, this study provides insights into how phytoplankton communities populate oceanic ecosystems, which is relevant to understanding their capacity to respond to ongoing climate change.

SulfAtlas, the sulfatase database: state of the art and new developments.

SulfAtlas (https://sulfatlas.sb-roscoff.fr/) is a knowledge-based resource dedicated to a sequence-based classification of sulfatases. Currently four sulfatase families exist (S1-S4) and the largest family (S1, formylglycine-dependent sulfatases) is divided into subfamilies by a phylogenetic approach, each subfamily corresponding to either a single characterized specificity (or few specificities in some cases) or to unknown substrates. Sequences are linked to their biochemical and structural information according to an expert scrutiny of the available literature. Database browsing was initially made possible both through a keyword search engine and a specific sequence similarity (BLAST) server. In this article, we will briefly summarize the experimental progresses in the sulfatase field in the last 6 years. To improve and speed up the (sub)family assignment of sulfatases in (meta)genomic data, we have developed a new, freely-accessible search engine using Hidden Markov model (HMM) for each (sub)family. This new tool (SulfAtlas HMM) is also a key part of the internal pipeline used to regularly update the database. SulfAtlas resource has indeed significantly grown since its creation in 2016, from 4550 sequences to 162 430 sequences in August 2022.

Insights into the potential for mutualistic and harmful host-microbe interactions affecting brown alga freshwater acclimation.

Microbes can modify their hosts' stress tolerance, thus potentially enhancing their ecological range. An example of such interactions is Ectocarpus subulatus, one of the few freshwater-tolerant brown algae. This tolerance is partially due to its (un)cultivated microbiome. We investigated this phenomenon by modifying the microbiome of laboratory-grown E. subulatus using mild antibiotic treatments, which affected its ability to grow in low salinity. Low salinity acclimation of these algal-bacterial associations was then compared. Salinity significantly impacted bacterial and viral gene expression, albeit in different ways across algal-bacterial communities. In contrast, gene expression of the host and metabolite profiles were affected almost exclusively in the freshwater-intolerant algal-bacterial communities. We found no evidence of bacterial protein production that would directly improve algal stress tolerance. However, vitamin K synthesis is one possible bacterial service missing specifically in freshwater-intolerant cultures in low salinity. In this condition, we also observed a relative increase in bacterial transcriptomic activity and the induction of microbial genes involved in the biosynthesis of the autoinducer AI-1, a quorum-sensing regulator. This could have resulted in dysbiosis by causing a shift in bacterial behaviour in the intolerant algal-bacterial community. Together, these results provide two promising hypotheses to be examined by future targeted experiments. Although they apply only to the specific study system, they offer an example of how bacteria may impact their host's stress response.

Global Phylogeography of Marine Synechococcus in Coastal Areas Reveals Strong Community Shifts.

Marine Synechococcus comprise a numerically and ecologically prominent phytoplankton group, playing a major role in both carbon cycling and trophic networks in all oceanic regions except in the polar oceans. Despite their high abundance in coastal areas, our knowledge of Synechococcus communities in these environments is based on only a few local studies. Here, we use the global metagenome data set of the Ocean Sampling Day (June 21st, 2014) to get a snapshot of the taxonomic composition of coastal Synechococcus communities worldwide, by recruitment on a reference database of 141 picocyanobacterial genomes, representative of the whole Prochlorococcus, Synechococcus, and Cyanobium diversity. This allowed us to unravel drastic community shifts over small to medium scale gradients of environmental factors, in particular along European coasts. The combined analysis of the phylogeography of natural populations and the thermophysiological characterization of eight strains, representative of the four major Synechococcus lineages (clades I to IV), also brought novel insights about the differential niche partitioning of clades I and IV, which most often co-dominate the Synechococcus community in cold and temperate coastal areas. Altogether, this study reveals several important characteristics and specificities of the coastal communities of Synechococcus worldwide. IMPORTANCE Synechococcus is the second most abundant phytoplanktonic organism on Earth, and its wide genetic diversity allowed it to colonize all the oceans except for polar waters, with different clades colonizing distinct oceanic niches. In recent years, the use of global metagenomics data sets has greatly improved our knowledge of "who is where" by describing the distribution of Synechococcus clades or ecotypes in the open ocean. However, little is known about the global distribution of Synechococcus ecotypes in coastal areas, where Synechococcus is often the dominant phytoplanktonic organism. Here, we leverage the global Ocean Sampling Day metagenomics data set to describe Synechococcus community composition in coastal areas worldwide, revealing striking community shifts, in particular along the coasts of Europe. As temperature appears as an important driver of the community composition, we also characterize the thermal preferenda of 8 Synechococcus strains, bringing new insights into the adaptation to temperature of the dominant Synechococcus clades.

Genomic patterns of divergence in the early and late steps of speciation of the deep-sea vent thermophilic worms of the genus Alvinella.

BACKGROUND: The transient and fragmented nature of the deep-sea hydrothermal environment made of ridge subduction, plate collision and the emergence of new rifts is currently acting to separate of vent populations, promoting local adaptation and contributing to bursts of speciation and species specialization. The tube-dwelling worms Alvinella pompejana called the Pompeii worm and its sister species A. caudata live syntopically on the hottest part of deep-sea hydrothermal chimneys along the East Pacific Rise. They are exposed to extreme thermal and chemical gradients, which vary greatly in space and time, and thus represent ideal candidates for understanding the evolutionary mechanisms at play in the vent fauna evolution. RESULTS: We explored genomic patterns of divergence in the early and late stages of speciation of these emblematic worms using transcriptome assemblies and the first draft genome to better understand the relative role of geographic isolation and habitat preference in their genome evolution. Analyses were conducted on allopatric populations of Alvinella pompejana (early stage of separation) and between A. pompejana and its syntopic species Alvinella caudata (late stage of speciation). We first identified divergent genomic regions and targets of selection as well as their position in the genome over collections of orthologous genes and, then, described the speciation dynamics by documenting the annotation of the most divergent and/or positively selected genes involved in the isolation process. Gene mapping clearly indicated that divergent genes associated with the early stage of speciation, although accounting for nearly 30% of genes, are highly scattered in the genome without any island of divergence and not involved in gamete recognition or mito-nuclear incompatibilities. By contrast, genomes of A. pompejana and A. caudata are clearly separated with nearly all genes (96%) exhibiting high divergence. This congealing effect however seems to be linked to habitat specialization and still allows positive selection on genes involved in gamete recognition, as a possible long-duration process of species reinforcement. CONCLUSION: Our analyses highlight the non-negligible role of natural selection on both the early and late stages of speciation in the iconic thermophilic worms living on the walls of deep-sea hydrothermal chimneys. They shed light on the evolution of gene divergence during the process of speciation and species specialization over a very long period of time.

Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Inter-Specific Genetic Exchange Despite Strong Divergence in Deep-Sea Hydrothermal Vent Gastropods of the Genus Alviniconcha.

Deep hydrothermal vents are highly fragmented and unstable habitats at all temporal and spatial scales. Such environmental dynamics likely play a non-negligible role in speciation. Little is, however, known about the evolutionary processes that drive population-level differentiation and vent species isolation and, more specifically, how geography and habitat specialisation interplay in the species history of divergence. In this study, the species range and divergence of Alviniconcha snails that occupy active Western Pacific vent fields was assessed by using sequence variation data of the mitochondrial Cox1 gene, RNAseq, and ddRAD-seq. Combining morphological description and sequence datasets of the three species across five basins, we confirmed that A. kojimai, A. boucheti, and A. strummeri, while partially overlapping over their range, display high levels of divergence in the three genomic compartments analysed that usually encompass values retrieved for reproductively isolated species with divergences rang from 9% to 12.5% (mtDNA) and from 2% to 3.1% (nuDNA). Moreover, the three species can be distinguished on the basis of their external morphology by observing the distribution of bristles and the shape of the columella. According to this sampling, A. boucheti and A. kojimai form an east-to-west species abundance gradient, whereas A. strummeri is restricted to the Futuna Arc/Lau and North Fiji Basins. Surprisingly, population models with both gene flow and population size heterogeneities among genomes indicated that these three species are still able to exchange genes due to secondary contacts at some localities after a long period of isolation.

Comparative Thermophysiology of Marine Synechococcus CRD1 Strains Isolated From Different Thermal Niches in Iron-Depleted Areas.

Marine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade. Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III, and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.

Identification of a New Set of Polypeptidic Sex Pheromones from Cuttlefish (Sepia officinalis).

The common English Channel cuttlefish (Sepia officinalis) reproduces every year on very localized coastal spawning areas after a west-east horizontal migration of several tens of kilometers (80-200 km). The massive arrival of spawners on the coasts of west Cotentin and the Bay of Seine is suspected to be driven by the action of sex pheromones expressed and secreted by the genitals of sexually mature females. The present study aims to verify the existence of polypeptide pheromones, of a higher molecular weight than those described above. Their size could confer them a wider range of action than that of the previously identified peptide pheromones. The implementation of an experimental strategy combining transcriptomics and proteomics with functional tests and an in silico study led to the identification of a cocktail of pheromones with molecular weights ranging between 22 and 26 kDa. Proteomic analyses combined to functional tests revealed partial pheromone release in the environment, and their accumulation in the outer capsule of the egg, suggesting the eggs as pheromone diffusers, also able to induce stimulation by contact when the eggs are handled by females.

metaGOflow: a workflow for the analysis of marine Genomic Observatories shotgun metagenomics data.

BACKGROUND: Genomic Observatories (GOs) are sites of long-term scientific study that undertake regular assessments of the genomic biodiversity. The European Marine Omics Biodiversity Observation Network (EMO BON) is a network of GOs that conduct regular biological community samplings to generate environmental and metagenomic data of microbial communities from designated marine stations around Europe. The development of an effective workflow is essential for the analysis of the EMO BON metagenomic data in a timely and reproducible manner. FINDINGS: Based on the established MGnify resource, we developed metaGOflow. metaGOflow supports the fast inference of taxonomic profiles from GO-derived data based on ribosomal RNA genes and their functional annotation using the raw reads. Thanks to the Research Object Crate packaging, relevant metadata about the sample under study, and the details of the bioinformatics analysis it has been subjected to, are inherited to the data product while its modular implementation allows running the workflow partially. The analysis of 2 EMO BON samples and 1 Tara Oceans sample was performed as a use case. CONCLUSIONS: metaGOflow is an efficient and robust workflow that scales to the needs of projects producing big metagenomic data such as EMO BON. It highlights how containerization technologies along with modern workflow languages and metadata package approaches can support the needs of researchers when dealing with ever-increasing volumes of biological data. Despite being initially oriented to address the needs of EMO BON, metaGOflow is a flexible and easy-to-use workflow that can be broadly used for one-sample-at-a-time analysis of shotgun metagenomics data.

Marine Synechococcus picocyanobacteria: Light utilization across latitudes.

The most ubiquitous cyanobacteria, Synechococcus, have colonized different marine thermal niches through the evolutionary specialization of lineages adapted to different ranges of temperature seawater. We used the strains of Synechococcus temperature ecotypes to study how light utilization has evolved in the function of temperature. The tropical Synechococcus (clade II) was unable to grow under 16 °C but, at temperatures >25 °C, induced very high growth rates that relied on a strong synthesis of the components of the photosynthetic machinery, leading to a large increase in photosystem cross-section and electron flux. By contrast, the Synechococcus adapted to subpolar habitats (clade I) grew more slowly but was able to cope with temperatures <10 °C. We show that growth at such temperatures was accompanied by a large increase of the photoprotection capacities using the orange carotenoid protein (OCP). Metagenomic analyzes revealed that Synechococcus natural communities show the highest prevalence of the ocp genes in low-temperature niches, whereas most tropical clade II Synechococcus have lost the gene. Moreover, bioinformatic analyzes suggested that the OCP variants of the two cold-adapted Synechococcus clades I and IV have undergone evolutionary convergence through the adaptation of the molecular flexibility. Our study points to an important role of temperature in the evolution of the OCP. We, furthermore, discuss the implications of the different metabolic cost of these physiological strategies on the competitiveness of Synechococcus in a warming ocean. This study can help improve the current hypotheses and models aimed at predicting the changes in ocean carbon fluxes in response to global warming.

Marine Transcriptomics Analysis for the Identification of New Antimicrobial Peptides.

Antimicrobial peptides (AMPs) participate in the immune system to avoid infection, are present in all living organisms and can be used as drugs. Fish express numerous AMP families including defensins, cathelicidins, liver-expressed antimicrobial peptides (LEAPs), histone-derived peptides, and piscidins (a fish-specific AMP family). The present study demonstrates for the first time the occurrence of several AMPs in lionfish (Pterois volitans). Using the lionfish transcriptome, we identified four transcript sequences encoding cysteine-rich AMPs and two new transcripts encoding piscidin-like peptides. These AMPs are described for the first time in a species of the Scorpaenidae family. A functional approach on new pteroicidins was carried out to determine antimicrobial sequences and potential uses, with a view to using some of these AMPs for human health or in aquaculture.

Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov., two flavobacteria from the epiphytic microbiota of the brown alga Ascophyllum nodosum, and emended description of the genus Zobellia.

Four marine bacterial strains were isolated from a thallus of the brown alga Ascophyllum nodosum collected in Roscoff, France. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, gliding, rod-shaped and grew optimally at 25-30 °C, at pH 7-8 and with 2-4 % NaCl. Phylogenetic analyses of their 16S rRNA gene sequences showed that the bacteria were affiliated to the genus Zobellia (family Flavobacteriaceae, phylum Bacteroidetes). The four strains exhibited 97.8-100 % 16S rRNA gene sequence similarity values among themselves, 97.9-99.1 % to the type strains of Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T, and less than 99 % to other species of the genus Zobellia. The DNA G+C content of the four strains ranged from 36.7 to 37.7 mol%. Average nucleotide identity and digital DNA-DNA hybridization calculations between the new strains and other members of the genus Zobellia resulted in values of 76.4-88.9 % and below 38.5 %, respectively. Phenotypic, phylogenetic and genomic analyses showed that the four strains are distinct from species of the genus Zobellia with validly published names. They represent two novel species of the genus Zobellia, for which the names Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov. are proposed with Asnod1-F08T (RCC6906T=KMM 6823T=CIP 111902T) and Asnod2-B07-BT (RCC6908T=KMM 6825T=CIP 111904T), respectively, as the type strains.

High-throughput sequencing on preservative ethanol is effective at jointly examining infraspecific and taxonomic diversity, although bioinformatics pipelines do not perform equally.

High-throughput sequencing of amplicons (HTSA) has been proposed as an effective approach to evaluate taxonomic and genetic diversity at the same time. However, there are still uncertainties as to how the results produced by different bioinformatics treatments impact the conclusions drawn on biodiversity and population genetics indices.We evaluated the ability of six bioinformatics pipelines to recover taxonomic and genetic diversity from HTSA data obtained from controlled assemblages. To that end, 20 assemblages were produced using 354 colonies of Botrylloides spp., sampled in the wild in ten marinas around Brittany (France). We used DNA extracted from preservative ethanol (ebDNA) after various time of storage (3, 6, and 12 months), and from a bulk of preserved specimens (bulkDNA). DNA was amplified with primers designed for targeting this ascidian genus. Results obtained from HTSA data were compared with Sanger sequencing on individual zooids (i.e., individual barcoding).Species identification and relative abundance determined with HTSA data from either ebDNA or bulkDNA were similar to those obtained with traditional individual barcoding. However, after 12 months of storage, the correlation between HTSA and individual-based data was lower than after shorter durations. The six bioinformatics pipelines were able to depict accurately the genetic diversity using standard population genetics indices (HS and FST), despite producing false positives and missing rare haplotypes. However, they did not perform equally and dada2 was the only pipeline able to retrieve all expected haplotypes.This study showed that ebDNA is a nondestructive alternative for both species identification and haplotype recovery, providing storage does not last more than 6 months before DNA extraction. Choosing the bioinformatics pipeline is a matter of compromise, aiming to retrieve all true haplotypes while avoiding false positives. We here recommend to process HTSA data using dada2, including a chimera-removal step. Even if the possibility to use multiplexed primer sets deserves further investigation to expand the taxonomic coverage in future similar studies, we showed that primers targeting a particular genus allowed to reliably analyze this genus within a complex community.

Robustness analysis of metabolic predictions in algal microbial communities based on different annotation pipelines.

Animals, plants, and algae rely on symbiotic microorganisms for their development and functioning. Genome sequencing and genomic analyses of these microorganisms provide opportunities to construct metabolic networks and to analyze the metabolism of the symbiotic communities they constitute. Genome-scale metabolic network reconstructions rest on information gained from genome annotation. As there are multiple annotation pipelines available, the question arises to what extent differences in annotation pipelines impact outcomes of these analyses. Here, we compare five commonly used pipelines (Prokka, MaGe, IMG, DFAST, RAST) from predicted annotation features (coding sequences, Enzyme Commission numbers, hypothetical proteins) to the metabolic network-based analysis of symbiotic communities (biochemical reactions, producible compounds, and selection of minimal complementary bacterial communities). While Prokka and IMG produced the most extensive networks, RAST and DFAST networks produced the fewest false positives and the most connected networks with the fewest dead-end metabolites. Our results underline differences between the outputs of the tested pipelines at all examined levels, with small differences in the draft metabolic networks resulting in the selection of different microbial consortia to expand the metabolic capabilities of the algal host. However, the consortia generated yielded similar predicted producible compounds and could therefore be considered functionally interchangeable. This contrast between selected communities and community functions depending on the annotation pipeline needs to be taken into consideration when interpreting the results of metabolic complementarity analyses. In the future, experimental validation of bioinformatic predictions will likely be crucial to both evaluate and refine the pipelines and needs to be coupled with increased efforts to expand and improve annotations in reference databases.