Bacterial lifestyle switch in response to algal metabolites

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we profiled bacterial transcriptomes in response to infochemicals derived from algae in exponential and stationary growth, which induced the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. We found that algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. In the pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and many transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7, and negated the DMSP-inducing lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological status of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during the interactions.

Unravelling microalgal-bacterial interactions in aquatic ecosystems through 16S co-occurrence networks

Microalgae and bacteria have a wide spectrum of associations in aquatic environments. Since their interactions can directly influence global carbon and nutrient cycling, understanding these associations help us evaluate their influence on ecosystem productivity. Algal biodiversity is large, and bacterial associations have been characterised for a small fraction of them. While experiments based on algal-bacterial co-culturing are commonly used to infer interactions, deciphering all associations present in nature through such methods is impractical and approaches based on co-occurrence network analysis can help infer associations. In this study, we used microbial co-occurrence networks built from Earth microbiome project 16S metabarcoding data to detect microalgal-bacterial associations in aquatic environments. We analysed marine and freshwater environments to understand what groups of bacteria are tightly co-occurring with different algal groups in both aquatic environments, to see patterns of interactions, and to evaluate the overall use of co-occurrence networks to infer meaningful algal-bacterial interactions. In line with expectations from co-culturing work, our results show that the phyla Proteobacteria and Bacteroidetes are the major bacterial associates of microalgae and the co-occurring bacteria may be specific to the algal host. From the independent analysis of environments, we also show that sample origin may be an important determinant of these interactions. By unravelling previously established microalgal-bacterial links as well as identifying a range of previously unknown interactions, we show that co-occurrence network analysis is a promising hypothesis-generating framework to study microalgal-bacterial interactions that can guide future research into the functional nature of interactions.

Physiological and Dual Transcriptional Analysis of Microalga Graesiella emersonii–Amoeboaphelidium protococcarum Pathosystem Uncovers Conserved Defense Response and Robust Pathogenicity

The underlying mechanisms of microalgal host–pathogen interactions remain largely unknown. In this study, we applied physiological and simultaneous dual transcriptomic analysis to characterize the microalga Graesiella emersonii–Amoeboaphelidium protococcarum interaction. Three infection stages were determined according to infection rate and physiological features. Dual RNA-seq results showed that the genes expression of G. emersonii and A. protococcarum were strongly dynamically regulated during the infection. For microalgal hosts, similar to plant defense response, the expression of defense genes involved in the pattern recognition receptors, large heat shock proteins, and reactive oxygen scavenging enzymes (glutathione, ferritin, and catalase) were significantly upregulated during infection. However, some genes encoding resistance proteins (R proteins) with a leucine-rich repeat domain exhibited no significant changes during infection. For endoparasite A. protococcarum, genes for carbohydrate-active enzymes, pathogen–host interactions, and putative effectors were significantly upregulated during infection. Furthermore, the genes in cluster II were significantly enriched in pathways associated with the modulation of vacuole transport, including endocytosis, phagosome, ubiquitin-mediated proteolysis, and SNARE interactions in vesicular transport pathways. These results suggest that G. emersonii has a conserved defense system against pathogen and that endoparasite A. protococcarum possesses a robust pathogenicity to infect the host. Our study characterizes the first transcriptomic profile of microalgae–endoparasite interaction, providing a new promising basis for complete understanding of the algal host defense strategies and parasite pathogenicity.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community development is influenced by spatial proximity to their algal host. Here we introduce a copolymer-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate, we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the porous structure. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

Intracellular development and impact of a eukaryotic parasite on its zombified microalgal host in the marine plankton

Parasites are widespread and diverse in the oceanic plankton, and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and whether the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the parasite Amoebophrya (Syndiniales) during intracellular infection (e.g. 200-fold increase of mitochondrion volume), particularly following digestion of nutrient-rich host chromosomes. Some of these changes are also found in the apicomplexan parasites (e.g. sequential acristate and cristate mitochondrion, switch from glycolysis to TCA), thus underlining key evolutionary-conserved mechanisms. In the algal host, energy-producing organelles (chloroplast) remain intact during most of the infection, but sugar reserves diminish while lipid droplets increase. Thus, rapid infection of the host nucleus could be a zombifying strategy to digest nutrient-rich chromosomes and escape cytoplasmic defense while benefiting from the maintained C-energy production of the host cell.

Physiological and Dual Transcriptional Analysis of Microalga Graesiella Emersonii–Amoeboaphelidium Protococcarum Pathosystem Uncovers Conserved Defense Response and Robust Pathogenicity

Background: The oleaginous microalga Graesiella emersonii, a potential industrial strain for lipid production, is frequently infected by the endoparasite Amoeboaphelidium protococcarum. It is essential to investigate the microalgae–endoparasite interaction to prevent and control microalgal diseases. However, the underlying mechanisms of microalgal host-pathogen interactions remain largely unknown. In this study, we applied physiological and simultaneous dual transcriptomic analysis to characterize the G. emersonii–A. protococcarum interaction for the first time. Results: Three infection stages were determined according to infection rate and physiological features. Dual RNA-seq results showed that the genes expression of G. emersonii and A. protococcarum were strongly dynamically regulated during the infection. For microalgal hosts, the expression of defense genes involved in the pattern recognition receptors, large heat shock proteins, and reactive oxygen scavenging enzymes (glutathione, ferritin, and catalase) were significantly upregulated during infection. However, some genes encoding resistance proteins (R proteins) with a leucine-rich repeat domain exhibited no significant changes during infection. Furthermore, ubiquitin-mediated proteolysis and endocytosis were strongly affected by A. protococcarum infection. For endoparasite A. protococcarum, genes for carbohydrate-active enzymes, pathogen-host interactions, and putative effectors were significantly upregulated during infection. Furthermore, the genes in cluster II were significantly enriched in pathways associated with the modulation of vacuole transport, including endocytosis, phagosome, ubiquitin-mediated proteolysis, and SNARE interactions in vesicular transport pathways.Conclusions: Our data provide a new promising basis for complete understanding of the algal host defense strategies and parasite pathogenicity. This is beneficial for the screening of resistant microalgal strains and developing control strategies for microalgal diseases.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community is developed by spatial proximity to their algal host. Here we introduce a hydrogel-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the hydrogel. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

Comparative genomics reveals insights into phylogenomic taxonomy and potential algae-bacteria interactions of novel versatile Mameliella alba strain LZ-28 isolated from highly-toxic marine phycosphere

Phycosphere harbors cross-kingdom interactions with significant ecological relevance for harmful algal blooms (HAB) and phycotoxins biosynthesis. Previously, a new red-pigmented bacterium designated as strain LZ-28 was isolated from phycosphere microbiota of typical HAB dinoflagellate Alexandrium catenella LZT09 which is a vitamin B 12 auxotroph and produces high levels of paralytic shellfish poisoning toxins (PST). Strain LZ-28 exhibited obvious growth-promoting activity toward its algal host, along with the production of active bioflocculanting exopolysaccharides (EPS). But the phylogenetic affiliation and genomic potential of this versatile bacterium has not yet been elucidated. In this study, we carried out combined taxonomic and phylogenomic analysis to clarify the taxonomic classification of strain LZ-28. The obtained 16S rRNA phylogeny revealed close taxonomic relationship between strain LZ-28 and other Mameliella alba members. Additional calculations of key phylogenomic parameters, average nucleotide identity (ANI), the average amino acid identity (AAI) and the digital DNA-DNA hybridization (dDDH) values based on genomes of strain LZ-28 and type strain of Mameliella alba were all exceeded the limit of species circumscription. Collectively considering the phenotypic and biochemical characterizations, strain LZ-28 was therefore identified as a new member of Mameliella alba. Furthermore, based on the genomic evidence, potential algae-bacteria interactions of strain LZ-28 with host algae LZT09 were elucidated through the associations with photosynthetic and antioxidant carotenoids, supplying of bacterial VB 12 to auxotroph host, and versatile EPS serving for bacterial colonization and nutrient exchange during their interactions, along with stress response systems to defense oxidative stress and quorum sensing (QS) signals benefited survival for bacteria in the symbiotic system. Comparative genomics shed light on similar genomic features between M . alba strains, revealed potential close associations of strain LZ-28 with its algae host, and further enriched the genomic repertoire of interactions between phycosphere microbiota and algal host LZT09.

Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.

Changing microbial activities during low salinity acclimation in the brown alga Ectocarpus subulatus

SummaryEctocarpus subulatus is one of the few brown algae found in river habitats. Its ability to tolerate freshwater is due, in part, to its uncultivated 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. The acclimation to low salinity of fresh water-tolerant and intolerant holobionts was then compared. Salinity had a significant impact on bacterial gene expression as well as the expression of algae- and bacteria-associated viruses in all holobionts, albeit in different ways for each holobiont. On the other hand, gene expression of the algal host and metabolite profiles were affected almost exclusively in the fresh water intolerant holobiont. We found no evidence of bacterial protein production that would directly improve algal stress tolerance. However, we identified vitamin K synthesis as one possible bacterial service missing specifically in the fresh water-intolerant holobiont in low salinity.We also noticed an increase in bacterial transcriptomic activity and the induction of microbial genes involved in the biosynthesis of the autoinducer AI-1, a compound that regulates quorum sensing. This could have caused a shift in bacterial behavior in the intolerant holobiont, resulting in virulence or dysbiosis.Originality-Significance StatementThe importance of symbiotic microbes for the health and stress resistance of multicellular eukaryotes is widely acknowledged, but understanding the mechanisms underlying these interactions is challenging. They are especially difficult to separate in systems with one or more uncultivable components. We bridge the gap between fully controlled, cultivable model systems and purely environmental studies through the use of a multi-omics approach and metabolic models on experimentally modified “holobiont” systems. This allows us to generate two promising working hypotheses on the mechanisms by which uncultivated bacteria influence their brown algal host’s fresh water tolerance.