Environmental Microbiology

Bacterial extracellular vesicles (BEVs) are nanoscale membranous structures released by diverse types of bacteria. Laboratory model systems indicate that these nanoparticles may play several roles in the ecophysiology of marine bacteria. However, their actual functionality in the environment remains unclear. Here we describe the proteomic composition of marine BEVs over more than 5,000 nautical miles of surface waters in the South Pacific, linking BEV cargoes to the bacterial communities producing them. BEVs were consistently present across a range of biogeochemical conditions, with an overall abundance comparable to that of bacterial cells. However, the protein content of the BEVs varied significantly between different ocean regions. The BEVs were enriched in carbohydrate transporters under phytoplankton bloom conditions, and contained iron and phosphate uptake-related proteins in nutrient-limited waters. This suggests that BEVs could enable cells to perform key extracellular functions in the marine environment. Our observations further highlight the prevalence of BEVs and the biogeographic patterns of their functional potential across oceanic scales.

The symbioses between the vestimentiferan tubeworms and their chemosynthetic partners (Gammaproteobacteria, Chromatiales, Sedimenticolaceae) hallmark the success of these organisms in hydrothermal vent and hydrocarbon seep deep-sea habitats. The fidelity of these associations varies, as both the hosts and the symbionts can be loose in partner choice. Some tubeworms may host distinct symbiont phylotypes, which often co-occur in a single host individual. To better understand the genetic basis for the promiscuity of tubeworm symbioses, we curated and investigated metagenome-assembled genomes of two symbiont phylotypes (species, based on the average nucleotide identity <95%) in Lamellibrachia anaximandri, a vestimentiferan endemic to the Mediterranean Sea, in individuals collected from Palinuro hydrothermal vents (Italy) and hydrocarbon seeps (Eratosthenes seamount and Palmahim disturbance). Using comparative genomics, we show that mainly mobilome and defense mechanism-related features distinguish the symbiont genotypes. While many central metabolic functions are conserved in the tubeworm symbionts, nitrate respiration (Nar, Nap and Nas proteins) is modular, yet this modularity is not linked to speciation, but rather to local adaptation. Our results hint that variation in a single moonlighting protein may be responsible for the host-symbiont fidelity.

The hydrothermal vent tube worm Riftia pachyptila lives in intimate symbiosis with intracellular sulfur-oxidizing gammaproteobacteria. Although the symbiont population consists of a single 16S rRNA phylotype, bacteria in the same host animal exhibit a remarkable degree of metabolic diversity: They simultaneously utilize two carbon fixation pathways and various energy sources and electron acceptors. Whether these multiple metabolic routes are employed in the same symbiont cells, or rather in distinct symbiont subpopulations, was unclear. As Riftia symbionts vary considerably in cell size and shape, we enriched individual symbiont cell sizes by density gradient centrifugation in order to test whether symbiont cells of different sizes show different metabolic profiles. Metaproteomic analysis and statistical evaluation using clustering and random forests, supported by microscopy and flow cytometry, strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. Large symbionts, on the other hand, apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis in large symbionts indicates that in this late differentiation stage the symbionts metabolism is efficiently geared towards the production of organic material. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

The supply of clean water for human consumption is being challenged by the appearance of pesticide pollutants in groundwater ecosystems. Biological rapid sand filtration is a commonly employed method for the removal of organic and inorganic impurities in water which relies on the degradative properties of microorganisms for the removal of diverse contaminants, including pesticides. Although sustainable and relatively inexpensive, the bioremediation capabilities of rapid sand filters vary greatly across waterworks. Bioaugmentation efforts with degradation-proficient bacteria have proven difficult due to the inability of the exogenous microbes to stably colonize the sand filters. Pesticide degrading genes, however, are often encoded naturally by plasmids--extrachromosomal DNA elements that can transfer between bacteria--yet their ability to spread within rapid sand filters have remained unknown. To evaluate the potential use of plasmids for the dissemination of pesticide degrading genes, we examined the permissiveness of rapid sand filter communities towards four environmental transmissible plasmids; RP4, RSF1010, pKJK5 and TOL (pWWO), using a dual-fluorescent bioreporter platform combined with FACS and 16S rRNA gene amplicon sequencing. Our results reveal that plasmids can transfer at high frequencies and across distantly related taxa from rapid sand filter communities, emphasizing their suitability for introducing pesticide degrading determinants in the microbiomes of underperforming water purification plants.

Sulfur oxidation is an essential biogeochemical process in coastal sediments. Of the two typical sulfur-oxidizing metabolic pathways, the reverse dissimilatory sulfite reductase pathway (rDsr) is recognized for its superior energy conservation efficiency compared to the Sox multi-enzyme system and thus might be important in intertidal habitats where electron acceptors are periodically limited. Here, we isolated 10 intertidal sediment strains that compose a novel Ruegeria lineage in the globally abundant marine Roseobacter group. They possess a complete rDsr pathway but lack the entire Sox system, whereas all known sulfur-oxidizing Roseobacters rely on Sox. In fact, rDsr-carrying but Sox-free bacteria are rare among all sequenced bacterial genomes (only 20 out of 35798), and how sulfur oxidation is driven exclusively by rDsr has not been investigated. Physiological assays, sulfur intermediate measurements, and transcriptomic analyses reveal that rDsr activation in this Ruegeria lineage occurs exclusively under microaerobic conditions and is coupled with thiosulfate oxidation in tandem with denitrification. Metagenomic analysis shows that rDsr sequences are enriched in marine habitats characterized by oxygen depletion, consistent with our physiological data. Further, the Rhodobacterales order where Ruegeria belongs accounts for approximately 5% of the rDsr-bearing bacterial community in intertidal sediments globally, underscoring its significant role in sulfur oxidation in this important marine environment.

Biological N2 fixation (BNF) can be traced to the Archean, over 3 Bya. The nitrogen isotopic fractionation composition ({delta}15N) of sedimentary rocks is commonly used to reconstruct the presence of diazotrophic ecosystems in the past. While {delta}15N has been calibrated under modern environmental conditions; it has not under Archean conditions, when atmospheric pO2 was lower and pCO2 was higher than today. Here we explore {delta}15N signatures in the laboratory under three simulated atmospheres with (i) elevated CO2 and no O2, (ii) present day CO2 and O2 and (iii) elevated CO2 and present day O2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set with literature data to examine for more generalized dependencies of {delta}15N during BNF across the Archaea and Bacteria, including cyanobacteria, and habitats. We find a mean {varepsilon}-value of -1.38 {+/-} 0.95, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set reveal correlations of isotopic fractionation of BNF with CO2 concentrations, toxin production and light, although within 1 {per thousand}. Moreover, correlation showed significant dependency of the magnitude of {varepsilon} to species type, C/N ratios and toxin production in heterocytous cyanobacteria, albeit it within a small range (-1.44 {+/-} 0.89). We therefore conclude that {delta}15N is likely robust when applied to the Archean, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower {varepsilon}) observed in the toxin producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation. ImportanceNitrogen is an essential element of life on Earth, however, despite its abundance it is not biologically accessible. Biological nitrogen fixation is an essential process whereby microbes fix N2 into biologically usable NH3. During this process, the enzyme nitrogenase preferentially uses light 14N, resulting in 15N depleted biomass. This signature can be traced back in time in sediments on Earth, and possibly other planets. In this paper, we explore the influence of pO2 and pCO2 on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO2, light and toxin producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin producing Nodularia and Nostoc species, that offers insight into why some organisms produce these N-rich toxic secondary metabolites.

Proliferations of toxic benthic cyanobacteria are increasingly being reported around the world. Of particular concern are Microcoleus-dominated mats associated with anatoxin production that have resulted in dog fatalities. Although the impact of cyanophages has been demonstrated in planktonic systems, their role in the population dynamics of benthic cyanobacteria has received little attention. Here we use metagenomics to explore phage presence in benthic mats from the Wolastoq|Saint John River (WR; New Brunswick, Canada) and Eel River (ER; California, US). Our survey recovered multiple viral-like sequences associated with different putative bacterial hosts, including two cyanophage genomes with apparently different replication strategies. A lysogenic cyanophage (predicted as a prophage) was found integrated in the genomes of Microcoleus sp. 3 recovered from five ER mat samples. This Microcoleus phage is related to previously described Phormidium phage counterparts. Also, we recovered lytic cyanophages from WR and ER mats dominated by anatoxin-producing Microcoleus, which was predicted as the putative host. Despite the geographical distance between WR and ER, the lytic Microcoleus phage genomes recovered from each river have similar sizes (circa 239 Kbp) and share similar gene content with high sequence identity. Phylogenetic analysis suggests that these lytic Microcoleus phages are distant from any other cyanophage previously described. Our results constitute the first report of cyanophages predicted to infect and therefore influence the population dynamics of mat-forming Microcoleus spp. associated with anatoxin production.

The degree of cyclization, or ring index (RI), in archaeal glycerol dibiphytanyl glycerol tetraether (GDGT) lipids was long thought to reflect homeoviscous adaptation to temperature. However, more recent experiments show that other factors (e.g., pH, growth phase, and energy flux) can also affect membrane composition. The main objective of this study was to investigate the effect of carbon and energy metabolism on membrane cyclization. To do so we cultivated Acidianus sp. DS80, a metabolically flexible and thermoacidophilic archaeon, on different electron donor, acceptor and carbon source combinations (S0/Fe3+/CO2, H2/Fe3+/CO2, H2/S0/CO2, or H2/S0/glucose). We show that differences in energy and carbon metabolism can result in over a full unit of change in RI in the thermoacidophile Acidianus sp. DS80. The patterns in RI correlated with the normalized electron transfer rate between electron donor and acceptor and did not always align with thermodynamic predictions of energy yield. In light of this, we discuss other factors that may affect the kinetics of cellular energy metabolism: electron transfer chain (ETC) efficiency, location of ETC reaction components (cytoplasmic vs. extracellular), and the physical state of electron donors and acceptors (gas vs. solid). Furthermore, assimilation of a more reduced form of carbon during heterotrophy appears to decrease the demand for reducing equivalents during lipid biosynthesis, resulting in lower RI. Together, these results point to the fundamental role of the cellular energy state in dictating GDGT cyclization, with those cells experiencing greater energy limitation synthesizing more cyclized GDGTs. ImportanceSome archaea make unique membrane-spanning lipids with different numbers of five or six membered rings in the core structure that modulate membrane fluidity and permeability. Changes in membrane core lipid composition reflect fundamental adaptation strategies of archaea in response to stress, but multiple environmental and physiological factors may affect the needs for membrane fluidity and permeability. In this study, we tested how Acidianus sp. DS80 changed its core lipid composition when grown with different electron donor/acceptor pairs. We show that changes in energy and carbon metabolisms significantly affected the relative abundance of rings in the core lipids of DS80. These observations highlight the need to better constrain metabolic parameters, in addition to environmental factors, that may influence changes in membrane physiology in Archaea. Such consideration would be particularly important for studying archaeal lipids from habitats that experience frequent environmental fluctuations and/or where metabolically diverse archaea thrive.

Molecular surveys of low temperature deep-sea hydrothermal vent fluids have shown that Campylobacteria (prev. Epsilonproteobacteria) often dominate the microbial community and that three subgroups - Arcobacter, Sulfurimonas and Sulfurovum - frequently coexist. In this study, we used replicated radiocarbon incubations of deep-sea hydrothermal fluids to investigate the activities of each group under three distinct incubation conditions. In order to quantify group-specific radiocarbon incorporation, we used newly designed oligonucleotide probes for Arcobacter, Sulfurimonas, and Sulfurovum to quantify their activity using catalyzed-reporter deposition fluorescence in-situ hybridization (CARD-FISH) combined with fluorescence-activated cell sorting. All three groups actively fixed CO2 in short-term (~ 20 h) incubations with either nitrate, oxygen, or no additions (control) at similar per-cell carbon fixation rates. Oxygen additions had the largest effect on community composition and overall cell numbers, and caused a pronounced shift in community composition at the amplicon sequence variant (ASV) level after only 20 h of incubation for all three groups. Interestingly, the effect of oxygen on carbon fixation rates appeared to depend on the initial starting community. Higher carbon fixation rates in oxygen-amended treatments were noted for all three taxa after an unintended disturbance to the sample site that may have selected for more oxygen-tolerant phylotypes. When viewed from a coarse taxonomic level, our data support assertions that these chemoautotrophic groups are functionally redundant in terms of their core metabolic capabilities since they were simultaneously active under all incubation conditions. In contrast, the higher resolution of amplicon sequencing allowed us to reveal finer-scale differences in growth that likely reflect adaptation of physiologically-distinct subtypes to varying oxygen concentrations in situ. Despite this progress, we still know remarkably little about the factors that maintain genomic diversity and allow for stable co-existence among these three campylobacterial groups. Moving forward, we suggest that more subtle biological factors such as enzyme substrate specificity, motility, cell morphology, and tolerance to environmental stress should be more thoroughly investigated to better understand ecological niche differentiation at deep-sea hydrothermal vents.

Temperature is a universal driver of microbial life, with rising sea surface temperatures expected to differentially influence the physiology, biodiversity, and distribution of bacteria and plankton. The impact of ocean warming on microbial interactions remains unclear, despite the importance of these relationships for ecosystem functioning. We employed weekly to monthly 18S and 16S rRNA gene amplicon metabarcoding over a full year (33 d) in a subtropical estuary, investigating microbial population dynamics and network interactions with respect to a temperature gradient (9-31{degrees}C). Certain microbes (e.g., Acidimicrobiia, Nitrososphaeria, and Syndiniales) increased in relative abundance with rising temperatures (Spearman {rho} > 0.69), whereas other groups (e.g., Alpha- and Gammaproteobacteria, Bacillariophyta, and Dinophyceae) slightly decreased, became saturated, or remained stable. With network analysis, we observed an increase in 18S- 18S interactions in warm (23-31{degrees}C) vs. cold (<23{degrees}C) temperatures, largely involving Syndiniales, Bacillariophyta, and Dinophyceae ASVs. Bacteria ASVs were more connected to other microbes (higher degree and centrality) and became more prominent in the cold network, highlighted by well-established cross-domain relationships (e.g., diatom-bacteria) and positive interactions among bacteria (e.g., SAR11 and Rhodobacterales). These efforts highlight the types of interactions that may be more common under changing temperatures, with implications for modeling biogeochemistry and assessing ecosystem health.

Nutrient starvation and subsequent mortality are processes that can shape ecosystem dynamics and influence global biogeochemical cycles yet are poorly understood. Here, we examined the dynamics of culture decline in 15 strains of Prochlorococcus, globally abundant marine cyanobacteria, under nitrogen (N) and phosphate (P) starvation. We then ask whether mortality patterns can be related to the evolutionary history of each strain, the geographic location and environmental conditions where it was isolated from, or the copy number of specific acquisition genes. We observed diverse decline patterns across starvation conditions and strains, identifying three differential features: maximum culture fluorescence, the number of fluorescence peaks during the decline stage, and the decline rate. Based on these features, each strain was categorized as being more sensitive to either nitrogen starvation or phosphorus/co-starvation. High light (HL) strains are more sensitive to N starvation, whereas other facets of the strains evolutionary or ecological origin were not correlated with mortality features. Surprisingly, the number of genes known to be involved in either N or P acquisition in each genome was not correlated with starvation sensitivity. Rather, genes involved in DNA damage repair were associated with N sensitivity to starvation, especially in HL strains, whereas genes related to protein quality control were more abundant in LL strains and associated with P/co starvation sensitivity. These findings reveal a previously unrecognized diversity in the dynamics of starvation and mortality across closely related Prochlorococcus strains, potentially driven by differences in the responses to DNA and protein damage.

The size structure of phytoplankton communities plays a key role in the fate of carbon fixed by photosynthesis. Whether phytoplankton cells sink, enter the microbial loop, or are consumed by larger organisms is generally determined by their size. Grazing has been advanced as a factor determining size structure, but sources of mortality other than grazing, such as viruses also are recognized to be important. Based on the observation that cell size and genome size are related in phytoplankton, we hypothesize that viruses can also play a role in shaping the size structure of the phytoplankton community. Because cell size is related to genome size, we suggest that phytoplankton species with larger genomes will have a more developed immune system to defend against viral infection. As a first step to test this hypothesis, we screened the published transcriptomes of 125 phytoplankton species for expressed viral and immune-response related genes. We found a significant negative correlation between host-cell size and viral-gene diversity, and a positive correlation between host-cell size and the number of immune-response related genes. Our hypothesis supported by preliminary findings opens new pathways to explore whether we should consider viruses as an additional evolutionary driver for larger phytoplankton size, along with grazing and nutrients.

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their co-occurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life-history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically, the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We gathered evidence for gene flow among co-occurring pathogenic and nonpathogenic strains, suggesting the potential of nonpathogenic strains to act as a reservoir of adaptive traits for pathogenic strains and vice versa. We further identified traits enriched in nonpathogens that suggest a strategy of stress tolerance, rather than avoidance, during their association with a broad range of host plants.

Bacteriophages (phages) are major drivers of microbial evolution and ecology, yet their diversity and functional roles remain poorly characterised in many natural environments, such as in freshwater systems. In boreal and subarctic freshwater habitats, where bacteria are typically slow-growing and nutrient-limited, phages are predicted to have a critical role in host regulation and horizontal gene exchange. However, only a few isolates have been obtained from such environments, leaving the genetic and functional diversity of these phages largely unexplored. Here, we present a collection of 40 bacteriophages isolated from boreal lakes and rivers using a set of diverse freshwater bacterial hosts. Despite using conventional isolation methods, eight of the isolates possess genomes larger than 200 kilobases and are classified as jumbophages. All jumbophages exhibited myovirus morphology and comparatively slow infection dynamics. These jumbophages include the first known representatives infecting members of Janthinobacterium and Herbaspirillum. Comparative genomic and phylogenetic analyses show that nearly all genomes are distinct from previously described phages, indicating substantial novelty. Diverse auxiliary metabolic and anti-defence systems were identified, including putative NAD+ salvage and acyl carrier protein modules, along with predicted Anti-Thoeris and Anti-CBASS elements. The Pseudomonas-infecting jumbophage Ahti encoded homologues of all 21 core genes that define the nucleus-forming family Chimalliviridae. Additionally, Ahti displayed compartmentalisation of DNA during infection, establishing it as the first freshwater nucleus-forming phage. These findings expand our understanding of the ecological, genomic, and functional diversity of phages in boreal environments, and highlight the role of freshwater ecosystems as significant reservoirs of novel viral lineages. ImportanceBacteriophages are viruses that infect bacteria and play important roles in shaping microbial communities and nutrient cycling in natural waters. However, much of what is known about their diversity comes from sequencing data alone, without environmental isolates that allow direct studies on their biology. In this study, we described 40 bacteriophages from boreal and subarctic lakes and rivers using freshwater bacterial hosts, including species of Flavobacterium, Pseudomonas, Janthinobacterium and other lesser-known phage hosts. Eight of these viruses had exceptionally large genomes, categorising them as jumbophages. These isolates included the first known jumbophages infecting Janthinobacterium and Herbaspirillum, as well as the first freshwater jumbophage shown to form a nucleus-like structure during infection. Together, these findings reveal boreal freshwaters as an important source of previously unknown virus diversity and provide new model systems for exploring phage biology in environmentally relevant contexts.

Bacterial membrane lipids have been traditionally defined as fatty acids (FAs) bilayers linked through ester bonds, while those of Archaea as ether-linked isoprenoids forming bilayers or monolayers of membrane spanning lipids (MSLs) known as isoprenoidal glycerol dialkyl glycerol tetraethers (isoGDGTs). This paradigm has been challenged with the discovery of branched GDGTs (brGDGTs), membrane spanning ether-bound branched alkyl FAs, that are of bacterial origin but whose specific producers in the environment are often unknown. The limited number of available microbial cultures restricts the knowledge of the biological sources of membrane lipids, which in turn limits their potential applicability as biomarkers. To address this limitation, we detected membrane lipids in the Black Sea using high resolution accurate mass/mass spectrometry and inferred their potential producers by targeting lipid biosynthetic pathways encoded on the metagenome, in metagenome-assembled genomes and unbinned scaffolds. We also detected brGDGTs and overly branched GDGTs in the suboxic and euxinic waters, which are potentially attributed, to members of the Planctomycetota, Cloacimonadota, Desulfobacterota, Chloroflexota, Actinobacteria and Myxococcota--all anaerobic microorganisms. These results open a new chapter in the use of specific brGDGTs as biomarkers of anoxic conditions in marine settings and of the role of these membrane lipids in microbial adaptation.

Despite the ecological importance of viruses, our understanding of their evolutionary dynamics in natural environments remains limited. This gap is particularly pronounced for giant dsDNA viruses of the phyla Nucleocytoviricota and Mirusviricota. Most knowledge on their population genetic dynamics is derived from a small number of laboratory-based experiments, whereas patterns in nature are rarely observed. To overcome this limitation, we traced genetic structure and transcription status of Heterosigma akashiwo virus (HaV) using high-frequency, time-resolved sampling during a host bloom in a coastal area of Japan, by integrating cell counting, metabarcoding, metagenomic and metatranscriptomic sequencing. Our study revealed that HaV dominated the giant virus community in most samples, with the relative abundances of up to 56%. Despite the high abundances, the HaV population exhibited a relatively low level of microdiversity but with a high pN/pS ratio compared to other giant viruses in the study site. Microdiversity increased during the early sampling period, reached a maximum at mid-sampling, and decreased during the later period, consistent with rapid diversification during viral expansion, possibly driven by both in situ mutations and the succession of pre-existing minor variants. Several accessory genes, including a glycosyltransferase and an endonuclease, were highly expressed, providing functional evidence consistent with host interaction-driven selective pressure during the bloom. Together, these results indicate that HaV population dynamics during algal blooms are shaped by host-driven selection acting on standing genetic variation.

Metolachlor (MET) is a widely used herbicide, and its extensive application has become a significant source of freshwater pollution. MET not only harms aquatic organisms but can also alter their ecological interactions, such as those between bloom-forming cyanobacteria and their chytrid parasites. Chytrids are zoosporic fungi that naturally regulate cyanobacterial populations. Although it is known that MET affects cyanobacterial metabolism and reduces chytrid fitness, the molecular mechanisms underlying these effects remain largely unknown. Here, we investigate the transcriptional profiles of the toxigenic bloom-forming cyanobacterium Planktothrix agardhii and its obligate chytrid parasite Rhizophydium megarrhizum exposed to an environmentally relevant concentration of MET. We found that MET alters the expression of genes associated with key physiological processes in both organisms. In P. agardhii, it affected genes involved in photosynthesis, gas vesicle synthesis and membrane stability. In R. megarrhizum, it influenced genes related to flagella assembly, developmental transitions and secondary metabolite production. Moreover, the distinct expression pattern observed under combined MET exposure and chytrid infection suggests a synergistic effect on the cyanobacterial host. By impairing chytrid-mediated control of cyanobacteria, MET may allow cyanobacteria to overgrow, thereby promoting bloom formation. This is the first study to uncover the genetic basis of herbicide-induced changes in cyanobacteria-fungal parasite dynamics.

Killer toxins are proteinaceous antifungal molecules produced by yeasts, with activity against a wide range of human and plant pathogenic fungi. Fungus gardens of attine ants in Brazil were surveyed to determine the presence of killer toxin-producing yeasts and to define their antifungal activities and ecological importance. Our results indicate that 10 out of 59 yeasts species isolated from fungal gardens are killer yeasts. Killer yeasts were less likely to inhibit the growth of yeasts isolated from the same environment but more effective at inhibiting yeast isolated from other environments, supporting a role for killer yeasts in shaping community composition. All killer yeasts harbored genome-encoded killer toxins lacked cytoplasmic toxin-encoding elements (i.e., double-stranded RNA satellites and linear double-stranded DNAs). Of all the killer yeasts associated with attine ants, Candida sinolaborantium (strain LESF 1467) showed a broad spectrum of antifungal activities against 39 out of 69 57% of yeast strains tested for toxin susceptibility. The complete genome sequence of C. sinolaborantium LESF 1467 identified a new killer toxin, Ksino, with similarities in primary sequence and tertiary structure to the Saccharomyces cerevisiae killer toxin named Klus. Surveys of publicly available genome databases identified homologs of Ksino in the genomes of yeast strains of Saccharomycetes and Pichiomycetes, as well as other species of Ascomycota and Basidiomycota filamentous fungi. This demonstrates that killer yeasts can be widespread in attine ant fungus gardens, possibly influencing fungal community composition and the importance of these complex microbial communities for discovering novel antifungal molecules. ImportanceAttine ants perform essential ecosystem services through the harvesting of substrates for fungiculture. The cultured fungi are a food source for attine ants. Characterizing antifungal toxin-producing yeasts (killer yeasts) is vital to understanding how they might protect gardens from invasion by unwanted fungal species. This study describes a new toxin named Ksino from the yeast Candida sinolaborantium, a member of a new group of toxins found across many different species of fungi. This work supports the role of killer yeasts in the ecology of fungicultures and competition between fungi. The observed high prevalence of killer yeasts in fungal gardens also enables the discovery of novel antifungal molecules with the potential to be applied against disease-causing fungi.

Coenocytic algae are organisms that undergo karyokinesis without cytokinesis, resulting in multinucleated cells. Most research on bacterial communities in coenocytic algae has used 16S rRNA sequencing, primarily focusing on the order Bryopsidales of green coenocytic algae. Recent studies have analyzed metagenome-assembled genomes (MAGs) from algal hosts across multiple taxa, such as Chlorophyta, Phaeophyta, and Rhodophyta, revealing more about bacterial biosynthetic machinery and potential symbiotic relationships. Given the cosmopolitan distribution of coenocytic algae, such as Bryopsis, Caulerpa, Codium, and the yellow-green alga Vaucheria, and their unique morphology, there is a need to better understand their associated bacterial communities. To address this, filaments of Vaucheria bursata LB2067 were sequenced using the Illumina NovaSeq instrument, and all publicly available short- and long-read datasets from coenocytic algae were screened for MAG recovery. All recovered MAGs from both Bryopsidales and Vaucheriales showed a high dominance of Pseudomonadota at the phylum level, but no consistent patterns at the order or family levels. High completeness of specific KEGG pathways, such as bidirectional polyphosphate metabolism and riboflavin biosynthesis, was prevalent in MAGs from coenocytic orders compared to non-coenocytic ones. Notably, N-acetylglutaminylglutamine amide (NAGGN) biosynthetic gene clusters (BGCs) were found only in MAGs from coenocytic orders, whereas polysaccharide utilization loci (PULs) were present in all MAGs analyzed. These results indicate that bacterial communities associated with coenocytic algae are complex, with diverse survival strategies adapted to challenging and variable environments.

Coral reefs are rapidly declining due to climate change, and natural recovery mechanisms can no longer keep pace. Coral probiotics have emerged as a promising restoration tool, yet their broad application is limited by our incomplete understanding of long-term microbial associations crucial to coral health. Microbes closely associated with the coral algal symbionts (Symbiodiniaceae) are particularly promising candidates, given their potential to enhance algal function and host resilience. Although recent studies have begun to characterize these Symbiodiniaceae-associated microbial communities, methodological differences in algal cell purification have led to inconsistent results. Here, we compared multiple sample processing steps to generate Symbiodiniaceae-enriched fractions from clonal fragments of Acropora nobilis, and examined the resulting microbial communities. We consistently detected members of Flavobacteraceae, Rhodobacteraceae, Rhizobiaceae, and the genus Marinobacter. Strikingly, Vibrio species dominated the Symbiodiniaceae fractions across protocols. We isolated and sequenced 11 Vibrio strains enriched in these fractions and identified genes related to both virulence and putative beneficial traits, including vitamin biosynthesis and antioxidant production. Despite confirming their high potential for virulence, the persistent association, spatial proximity to Symbiodiniaceae, and presence of genes suggesting beneficial functions point to possible mutualistic roles for these Vibrio strains within the coral holobiont. This work highlights the value of fraction-based sampling for resolving microbiome structure, and emphasizes the need to reassess the ecological roles of key microbial taxa. By advancing our understanding of Symbiodiniaceae-bacteria interactions, this work provides a foundation for exploring microbial contributions to coral health and resilience.