Ecophysiological and genomic approaches to cyanobacterial hardening for restoration.

Cyanobacteria inhabit extreme environments, including drylands, providing multiple benefits to the ecosystem. Soil degradation in warm drylands is increasing due to land use intensification. Restoration methods adapted to the extreme stress in drylands are being developed, such as cyanobacteria inoculation to recover biocrusts. For this type of restoration method to be a success, it is crucial to optimize the survival of inoculated cyanobacteria in the field. One strategy is to harden them to be acclimated to stressful conditions after laboratory culturing. Here, we analyzed the genome and ecophysiological response to osmotic desiccation and UVR stresses of an Antarctic cyanobacterium, Stenomitos frigidus ULC029, which is closely related to other cyanobacteria from warm and cold dryland soils. Chlorophyll a concentrations showed that preculturing ULC029 under moderate osmotic stress improved its survival during an assay of desiccation plus rehydration under UVR. Additionally, its sequential exposure to these stress factors increased the production of exopolysaccharides, carotenoids, and scytonemin. Desiccation, but not osmotic stress, increased the concentrations of the osmoprotectants trehalose and sucrose. However, osmotic stress might induce the production of other osmoprotectants, for which the complete pathways were observed in the ULC029 genome. In total, 140 genes known to be involved in stress resistance were annotated. Here, we confirm that the sequential application of moderate osmotic stress and dehydration could improve cyanobacterial hardening for soil restoration by inducing several resistance mechanisms. We provide a high-quality genome of ULC029 and a description of the main resistance mechanisms (i.e., production of exopolysaccharides, osmoprotectants, chlorophyll, and carotenoids; DNA repair; and oxidative stress protection).

Geology defines microbiome structure and composition in nunataks and valleys of the Sør Rondane Mountains, East Antarctica.

Understanding the relation between terrestrial microorganisms and edaphic factors in the Antarctic can provide insights into their potential response to environmental changes. Here we examined the composition of bacterial and micro-eukaryotic communities using amplicon sequencing of rRNA genes in 105 soil samples from the Sør Rondane Mountains (East Antarctica), differing in bedrock or substrate type and associated physicochemical conditions. Although the two most widespread taxa (Acidobacteriota and Chlorophyta) were relatively abundant in each sample, multivariate analysis and co-occurrence networks revealed pronounced differences in community structure depending on substrate type. In moraine substrates, Actinomycetota and Cercozoa were the most abundant bacterial and eukaryotic phyla, whereas on gneiss, granite and marble substrates, Cyanobacteriota and Metazoa were the dominant bacterial and eukaryotic taxa. However, at lower taxonomic level, a distinct differentiation was observed within the Cyanobacteriota phylum depending on substrate type, with granite being dominated by the Nostocaceae family and marble by the Chroococcidiopsaceae family. Surprisingly, metazoans were relatively abundant according to the 18S rRNA dataset, even in samples from the most arid sites, such as moraines in Austkampane and Widerøefjellet ("Dry Valley"). Overall, our study shows that different substrate types support distinct microbial communities, and that mineral soil diversity is a major determinant of terrestrial microbial diversity in inland Antarctic nunataks and valleys.

TerrANTALife 1.0 Biodiversity data checklist of known Antarctic terrestrial and freshwater life forms.

Background: Incomplete species inventories for Antarctica represent a key challenge for comprehensive ecological research and conservation in the region. Additionally, data required to understand population dynamics, rates of evolution, spatial ranges, functional traits, physiological tolerances and species interactions, all of which are fundamental to disentangle the different functional elements of Antarctic biodiversity, are mostly missing. However, much of the fauna, flora and microbiota in the emerged ice-free land of the continent have an uncertain presence and/or unresolved status, with entire biodiversity compendia of prokaryotic groups (e.g. bacteria) being missing. All the available biodiversity information requires consolidation, cross-validation, re-assessment and steady systematic inclusion in order to create a robust catalogue of biodiversity for the continent. New information: We compiled, completed and revised eukaryotic species inventories present in terrestrial and freshwater ecosystems in Antarctica in a new living database: terrANTALife (version 1.0). The database includes the first integration in a compendium for many groups of eukaryotic microorganisms. We also introduce a first catalogue of amplicon sequence variants (ASVs) of prokaryotic biodiversity. Available compendia and literature to date were searched for Antarctic terrestrial and freshwater species, integrated, taxonomically harmonised and curated by experts to create comprehensive checklists of Antarctic organisms. The final inventories comprises 470 animal species (including vertebrates, free-living invertebrates and parasites), 306 plants (including all Viridiplantae: embryophytes and green algae), 997 fungal species and 434 protists (sensu lato). We also provide a first account for many groups of microorganisms, including non-lichenised fungi and multiple groups of eukaryotic unicellular species (Stramenophila, Alveolata and Rhizaria (SAR), Chromists and Amoeba), jointly referred to as "protists". In addition, we identify 1753 bacterial (obtained from 348117 ASVs) and 34 archaeal genera (from 1848 ASVs), as well as, at least, 14 virus families. We formulate a basic tree of life in Antarctica with the main lineages listed in the region and their "known-accepted-species" numbers.

Polar lake microbiomes have distinct evolutionary histories.

Toward the poles, life on land is increasingly dominated by microorganisms, yet the evolutionary origin of polar microbiomes remains poorly understood. Here, we use metabarcoding of Arctic, sub-Antarctic, and Antarctic lacustrine benthic microbial communities to test the hypothesis that high-latitude microbiomes are recruited from a globally dispersing species pool through environmental selection. We demonstrate that taxonomic overlap between the regions is limited within most phyla, even at higher-order taxonomic levels, with unique deep-branching phylogenetic clades being present in each region. We show that local and regional taxon richness and net diversification rate of regionally restricted taxa differ substantially between polar regions in both microeukaryotic and bacterial biota. This suggests that long-term evolutionary divergence resulting from low interhemispheric dispersal and diversification in isolation has been a prominent process shaping present-day polar lake microbiomes. Our findings illuminate the distinctive biogeography of polar lake ecosystems and underscore that conservation efforts should include their unique microbiota.

Novel diversity of polar Cyanobacteria revealed by genome-resolved metagenomics.

Benthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into the diversity of polar Cyanobacteria, only a handful of genomes have been sequenced to date. Here, we applied a genome-resolved metagenomics approach to data obtained from Arctic, sub-Antarctic and Antarctic microbial mats. We recovered 37 metagenome-assembled genomes (MAGs) of Cyanobacteria representing 17 distinct species, most of which are only distantly related to genomes that have been sequenced so far. These include (i) lineages that are common in polar microbial mats such as the filamentous taxa Pseudanabaena, Leptolyngbya, Microcoleus/Tychonema and Phormidium; (ii) the less common taxa Crinalium and Chamaesiphon; (iii) an enigmatic Chroococcales lineage only distantly related to Microcystis; and (iv) an early branching lineage in the order Gloeobacterales that is distributed across the cold biosphere, for which we propose the name Candidatus Sivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria, especially in understudied remote and extreme environments.

Threat management priorities for conserving Antarctic biodiversity.

Antarctic terrestrial biodiversity faces multiple threats, from invasive species to climate change. Yet no large-scale assessments of threat management strategies exist. Applying a structured participatory approach, we demonstrate that existing conservation efforts are insufficient in a changing world, estimating that 65% (at best 37%, at worst 97%) of native terrestrial taxa and land-associated seabirds are likely to decline by 2100 under current trajectories. Emperor penguins are identified as the most vulnerable taxon, followed by other seabirds and dry soil nematodes. We find that implementing 10 key threat management strategies in parallel, at an estimated present-day equivalent annual cost of US$23 million, could benefit up to 84% of Antarctic taxa. Climate change is identified as the most pervasive threat to Antarctic biodiversity and influencing global policy to effectively limit climate change is the most beneficial conservation strategy. However, minimising impacts of human activities and improved planning and management of new infrastructure projects are cost-effective and will help to minimise regional threats. Simultaneous global and regional efforts are critical to secure Antarctic biodiversity for future generations.

LC-MS/MS Validation and Quantification of Cyanotoxins in Algal Food Supplements from the Belgium Market and Their Molecular Origins.

Food supplements are gaining popularity worldwide. However, harmful natural compounds can contaminate these products. In the case of algae-based products, the presence of toxin-producing cyanobacteria may cause health risks. However, data about the prevalence of algal food supplements on the Belgian market and possible contaminations with cyanotoxins are scarce. Therefore, we optimized and validated a method based on Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry to quantify eight microcystin congeners and nodularin in algal food supplements. Our analytical method was successfully validated and applied on 35 food supplement samples. Nine out of these samples contained microcystin congeners, of which three exceeded 1 µg g-1, a previously proposed guideline value. Additionally, the mcyE gene was amplified and sequenced in ten products to identify the taxon responsible for the toxin production. For seven out of these ten samples, the mcyE gene could be amplified and associated to Microcystis sp. EFSA and posology consumption data for algal-based food supplements were both combined with our toxin prevalence data to establish different toxin exposure scenarios to assess health risks and propose new guideline values.

Characterization of the Halochromic Gloeocapsin Pigment, a Cyanobacterial Biosignature for Paleobiology and Astrobiology.

Ultraviolet (UV)-screening compounds represent a substantial asset for the survival of cyanobacteria in extreme environments exposed to high doses of UV radiations on modern and early Earth. Among these molecules, the halochromic pigment gloeocapsin remains poorly characterized and studied. In this study, we identified a gloeocapsin-producing cultivable cyanobacteria: the strain Phormidesmis nigrescens ULC007. We succeeded to extract, to partially purify, and to compare the dark blue pigment from both the ULC007 culture and an environmental Gloeocapsa alpina dominated sample. FT-IR and Raman spectra of G. alpina and P. nigrescens ULC007 pigment extracts strongly suggested a common backbone structure. The high-pressure liquid chromatography-UV-MS/MS analysis of the ULC007 pigment extract allowed to narrow down the molecular formula of gloeocapsin to potentially five candidates within three classes of halochromic molecules: anthraquinone derivatives, coumarin derivatives, and flavonoids. With the discovery of gloeocapsin in P. nigrescens, the production of this pigment is now established for three lineages of cyanobacteria (including G. alpina, P. nigrescens, and Solentia paulocellulare) that belong to three distinct orders (Chroococcales, Pleurocapsales, Synechoccocales), inhabiting very diverse environments. This suggests that gloeocapsin production was a trait of their common ancestor or was acquired by lateral gene transfer. This work represents an important step toward the elucidation of the structure of this enigmatic pigment and its biosynthesis, and it potentially provides a new biosignature for ancient cyanobacteria. It also gives a glimpse on the evolution of UV protection strategies, which are relevant for early phototrophic life on Earth and possibly beyond.

Introducing Petrachlorosaceae fam. nov., Petrachloros gen. nov. and Petrachloros mirabilis sp. nov. (Synechococcales, Cyanobacteria) Isolated from a Portuguese UNESCO monument.

The Synechococcales is a large cyanobacterial order comprising both unicellular and filamentous forms, with parietal thylakoid arrangement. Previously, this order has been the subject of taxonomic revisions with new families being erected. During studies of the phototrophic communities on the limestone walls of the Old Cathedral of Coimbra (UNESCO monument), a coccoid Aphanocapsa-like cyanobacterium was isolated. It was characterized using a polyphasic approach, based on morphology, 16S rRNA phylogenetic and phylogenomic analyses, internal transcribed spacer (ITS) secondary structure, and ecology. The 16S rRNA phylogenetic analyses showed that this strain is placed in a separate and highly supported family-level clade, as part of a large group comprising the families Prochlorococcaceae and Prochlorotrichaceae, with Lagosinema as the closest (although quite distant) taxon. Additionally, the phylogenomic analysis also placed this strain in a separate lineage, situated distantly apart from the family Thermosynechococcaceae, but with strains assigned to Acaryochloris marina MBIC 11017 and Aphanocapsa montana BDHKU210001 as the closest taxa. Based on these data, as well as on the results from the secondary ITS structure, morphology, and ecology, we here propose the establishment of Petrachlorosaceae fam. nov., along with the description of Petrachloros gen. nov. and Petrachloros mirabilis sp. nov. We also address additional considerations regarding some cyanobacterial taxa within the order Synechococcales, which we believe deserve further revisions.

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations.

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L-1) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L-1 to 2798.81 µg L-1 total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.

The GEN-ERA toolbox: unified and reproducible workflows for research in microbial genomics.

BACKGROUND: Microbial culture collections play a key role in taxonomy by studying the diversity of their strains and providing well-characterized biological material to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (e.g., yeasts). FINDINGS: This public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenomic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling. TECHNICAL DETAILS: Nextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility. TESTING: The toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria. CONCLUSION: The GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be used by all researchers interested in microbial taxonomy, as exemplified by our case study on Gloeobacterales.

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

The continuous increase in sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects ever more complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow "ORPER", for "ORganism PlacER", containerized in Singularity, which allows the determination the phylogenetic position of a collection of organisms in the genomic landscape. ORPER constrains the phylogenetic placement of SSU (16S) rRNA sequences in a multilocus reference tree based on ribosomal protein genes extracted from public genomes. We demonstrate the utility of ORPER on the Cyanobacteria phylum, by placing 152 strains of the BCCM/ULC collection.

Cyanotoxins and Food Contamination in Developing Countries: Review of Their Types, Toxicity, Analysis, Occurrence and Mitigation Strategies.

Cyanotoxins have gained global public interest due to their potential to bioaccumulate in food, which threatens human health. Bloom formation is usually enhanced under Mediterranean, subtropical and tropical climates which are the dominant climate types in developing countries. In this context, we present an up-to-date overview of cyanotoxins (types, toxic effects, analysis, occurrence, and mitigation) with a special focus on their contamination in (sea)food from all the developing countries in Africa, Asia, and Latin America as this has received less attention. A total of 65 publications have been found (from 2000 until October 2021) reporting the contamination by one or more cyanotoxins in seafood and edible plants (five papers). Only Brazil and China conducted more research on cyanotoxin contamination in food in comparison to other countries. The majority of research focused on the detection of microcystins using different analytical methods. The detected levels mostly surpassed the provisional tolerable daily intake limit set by the World Health Organization, indicating a real risk to the exposed population. Assessment of cyanotoxin contamination in foods from developing countries still requires further investigations by conducting more survey studies, especially the simultaneous detection of multiple categories of cyanotoxins in food.

Cyanobacteria evolution: Insight from the fossil record.

Cyanobacteria played an important role in the evolution of Early Earth and the biosphere. They are responsible for the oxygenation of the atmosphere and oceans since the Great Oxidation Event around 2.4 Ga, debatably earlier. They are also major primary producers in past and present oceans, and the ancestors of the chloroplast. Nevertheless, the identification of cyanobacteria in the early fossil record remains ambiguous because the morphological criteria commonly used are not always reliable for microfossil interpretation. Recently, new biosignatures specific to cyanobacteria were proposed. Here, we review the classic and new cyanobacterial biosignatures. We also assess the reliability of the previously described cyanobacteria fossil record and the challenges of molecular approaches on modern cyanobacteria. Finally, we suggest possible new calibration points for molecular clocks, and strategies to improve our understanding of the timing and pattern of the evolution of cyanobacteria and oxygenic photosynthesis.

Marked Succession of Cyanobacterial Communities Following Glacier Retreat in the High Arctic.

Cyanobacteria are important colonizers of recently deglaciated proglacial soil but an in-depth investigation of cyanobacterial succession following glacier retreat has not yet been carried out. Here, we report on the successional trajectories of cyanobacterial communities in biological soil crusts (BSCs) along a 100-year deglaciation gradient in three glacier forefields in central Svalbard, High Arctic. Distance from the glacier terminus was used as a proxy for soil age (years since deglaciation), and cyanobacterial abundance and community composition were evaluated by epifluorescence microscopy and pyrosequencing of partial 16S rRNA gene sequences, respectively. Succession was characterized by a decrease in phylotype richness and a marked shift in community structure, resulting in a clear separation between early (10-20 years since deglaciation), mid (30-50 years), and late (80-100 years) communities. Changes in cyanobacterial community structure were mainly connected with soil age and associated shifts in soil chemical composition (mainly moisture, SOC, SMN, K, and Na concentrations). Phylotypes associated with early communities were related either to potentially novel lineages (< 97.5% similar to sequences currently available in GenBank) or lineages predominantly restricted to polar and alpine biotopes, suggesting that the initial colonization of proglacial soil is accomplished by cyanobacteria transported from nearby glacial environments. Late communities, on the other hand, included more widely distributed genotypes, which appear to establish only after the microenvironment has been modified by the pioneering taxa.

Metagenomic assembly of new (sub)polar Cyanobacteria and their associated microbiome from non-axenic cultures.

Cyanobacteria form one of the most diversified phyla of Bacteria. They are important ecologically as primary producers, for Earth evolution and biotechnological applications. Yet, Cyanobacteria are notably difficult to purify and grow axenically, and most strains in culture collections contain heterotrophic bacteria that were probably associated with Cyanobacteria in the environment. Obtaining cyanobacterial DNA without contaminant sequences is thus a challenging and time-consuming task. Here, we describe a metagenomic pipeline that enables the easy recovery of genomes from non-axenic cultures. We tested this pipeline on 17 cyanobacterial cultures from the BCCM/ULC public collection and generated novel genome sequences for 12 polar or subpolar strains and three temperate ones, including three early-branching organisms that will be useful for phylogenomics. In parallel, we assembled 31 co-cultivated bacteria (12 nearly complete) from the same cultures and showed that they mostly belong to Bacteroidetes and Proteobacteria, some of them being very closely related in spite of geographically distant sampling sites.

Consensus assessment of the contamination level of publicly available cyanobacterial genomes.

Publicly available genomes are crucial for phylogenetic and metagenomic studies, in which contaminating sequences can be the cause of major problems. This issue is expected to be especially important for Cyanobacteria because axenic strains are notoriously difficult to obtain and keep in culture. Yet, despite their great scientific interest, no data are currently available concerning the quality of publicly available cyanobacterial genomes. As reliably detecting contaminants is a complex task, we designed a pipeline combining six methods in a consensus strategy to assess the contamination level of 440 genome assemblies of Cyanobacteria. Two methods are based on published reference databases of ribosomal genes (SSU rRNA 16S and ribosomal proteins), one is indirectly based on a reference database of marker genes (CheckM), and three are based on complete genome analysis. Among those genome-wide methods, Kraken and DIAMOND blastx share the same reference database that we derived from Ensembl Bacteria, whereas CONCOCT does not require any reference database, instead relying on differences in DNA tetramer frequencies. Given that all the six methods appear to have their own strengths and limitations, we used the consensus of their rankings to infer that >5% of cyanobacterial genome assemblies are highly contaminated by foreign DNA (i.e., contaminants were detected by 5 or 6 methods). Our results will help researchers to check the quality of publicly available genomic data before use in their own analyses. Moreover, we argue that journals should make mandatory the submission of raw read data along with genome assemblies in order to facilitate the detection of contaminants in sequence databases.

A constrained SSU-rRNA phylogeny reveals the unsequenced diversity of photosynthetic Cyanobacteria (Oxyphotobacteria).

OBJECTIVE: Cyanobacteria are an ancient phylum of prokaryotes that contain the class Oxyphotobacteria. This group has been extensively studied by phylogenomics notably because it is widely accepted that Cyanobacteria were responsible for the spread of photosynthesis to the eukaryotic domain. The aim of this study was to evaluate the fraction of the oxyphotobacterial diversity for which sequenced genomes are available for genomic studies. For this, we built a phylogenomic-constrained SSU rRNA (16S) tree to pinpoint unexploited clusters of Oxyphotobacteria that should be targeted for future genome sequencing, so as to improve our understanding of Oxyphotobacteria evolution. RESULTS: We show that only a little fraction of the oxyphotobacterial diversity has been sequenced so far. Indeed 31 rRNA clusters of the 60 composing the photosynthetic Cyanobacteria have a fraction of sequenced genomes < 1%. This fraction remains low (min = 1%, median = 11.1%, IQR = 7.3%) within the remaining "sequenced" clusters that already contain some representative genomes. The "unsequenced" clusters are scattered across the whole Oxyphotobacteria tree, at the exception of very basal clades. Yet, these clades still feature some (sub)clusters without any representative genome. This last result is especially important, as these basal clades are prime candidate for plastid emergence.