Isolation, growth, and nitrogen fixation rates of the Hemiaulus-Richelia (diatom-cyanobacterium) symbiosis in culture

Nitrogen fixers (diazotrophs) are often an important nitrogen source to phytoplankton nutrient budgets in N-limited marine environments. Diazotrophic symbioses between cyanobacteria and diatoms can dominate nitrogen-fixation regionally, particularly in major river plumes and in open ocean mesoscale blooms. This study reports the successful isolation and growth in monocultures of multiple strains of a diatom-cyanobacteria symbiosis from the Gulf of Mexico using a modified artificial seawater medium. We document the influence of light and nutrients on nitrogen fixation and growth rates of the host diatom Hemiaulus hauckii Grunow together with its diazotrophic endosymbiont Richelia intracellularis Schmidt, as well as less complete results on the Hemiaulus membranaceus-R. intracellularis symbiosis. The symbioses rates reported here are for the joint diatom-cyanobacteria unit. Symbiont diazotrophy was sufficient to support both the host diatom and cyanobacteria symbionts, and the entire symbiosis replicated and grew without added nitrogen. Maximum growth rates of multiple strains of H. hauckii symbioses in N-free medium with N2 as the sole N source were 0.74–0.93 div d−1. Growth rates followed light saturation kinetics in H. hauckii symbioses with a growth compensation light intensity (EC) of 7–16 µmol m−2s−1and saturation light level (EK) of 84–110 µmol m−2s−1. Nitrogen fixation rates by the symbiont while within the host followed a diel pattern where rates increased from near-zero in the scotophase to a maximum 4–6 h into the photophase. At the onset of the scotophase, nitrogen-fixation rates declined over several hours to near-zero values. Nitrogen fixation also exhibited light saturation kinetics. Maximum N2 fixation rates (84 fmol N2 heterocyst−1h−1) in low light adapted cultures (50 µmol m−2s−1) were approximately 40–50% of rates (144–154 fmol N2 heterocyst−1h−1) in high light (150 and 200 µmol m−2s−1) adapted cultures. Maximum laboratory N2 fixation rates were ~6 to 8-fold higher than literature-derived field rates of the H. hauckii symbiosis. In contrast to published results on the Rhizosolenia-Richelia symbiosis, the H. hauckii symbiosis did not use nitrate when added, although ammonium was consumed by the H. hauckii symbiosis. Symbiont-free host cell cultures could not be established; however, a symbiont-free H. hauckii strain was isolated directly from the field and grown on a nitrate-based medium that would not support DDA growth. Our observations together with literature reports raise the possibility that the asymbiotic H. hauckii are lines distinct from an obligately symbiotic H. hauckii line. While brief descriptions of successful culture isolation have been published, this report provides the first detailed description of the approaches, handling, and methodologies used for successful culture of this marine symbiosis. These techniques should permit a more widespread laboratory availability of these important marine symbioses.

A microbial factory for defensive kahalalides in a tripartite marine symbiosis

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca. E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.

Origin of Chemical Diversity in Prochloron-Tunicate Symbiosis

ABSTRACTDiversity-generating metabolism leads to the evolution of many different chemicals in living organisms. Here, by examining a marine symbiosis, we provide a precise evolutionary model of how nature generates a family of novel chemicals, the cyanobactins. We show that tunicates and their symbioticProchloroncyanobacteria share congruent phylogenies, indicating thatProchloronphylogeny is related to host phylogeny and not to external habitat or geography. We observe thatProchloronexchanges discrete functional genetic modules for cyanobactin secondary metabolite biosynthesis in an otherwise conserved genetic background. The module exchange leads to gain or loss of discrete chemical functional groups. Because the underlying enzymes exhibit broad substrate tolerance, discrete exchange of substrates and enzymes betweenProchloronstrains leads to the rapid generation of chemical novelty. These results have implications in choosing biochemical pathways and enzymes for engineered or combinatorial biosynthesis.IMPORTANCEWhile most biosynthetic pathways lead to one or a few products, a subset of pathways are diversity generating and are capable of producing thousands to millions of derivatives. This property is highly useful in biotechnology since it enables biochemical or synthetic biological methods to create desired chemicals. A fundamental question has been how nature itself creates this chemical diversity. Here, by examining the symbiosis between coral reef animals and bacteria, we describe the genetic basis of chemical variation with unprecedented precision. New compounds from the cyanobactin family are created by either varying the substrate or importing needed enzymatic functions from other organisms or via both mechanisms. This natural process matches successful laboratory strategies to engineer the biosynthesis of new chemicals and teaches a new strategy to direct biosynthesis.

A New C-Type Lectin Similar to the Human Immunoreceptor DC-SIGN Mediates Symbiont Acquisition by a Marine Nematode

ABSTRACT Although thiotrophic symbioses have been intensively studied for the last three decades, nothing is known about the molecular mechanisms of symbiont acquisition. We used the symbiosis between the marine nematode Laxus oneistus and sulfur-oxidizing bacteria to study this process. In this association a monolayer of symbionts covers the whole cuticle of the nematode, except its anterior-most region. Here, we identify a novel Ca2+-dependent mannose-specific lectin that was exclusively secreted onto the posterior, bacterium-associated region of L. oneistus cuticle. A recombinant form of this lectin induced symbiont aggregation in seawater and was able to compete with the native lectin for symbiont binding in vivo. Surprisingly, the carbohydrate recognition domain of this mannose-binding protein was similar both structurally and functionally to a human dendritic cell-specific immunoreceptor. Our results provide a molecular link between bacterial symbionts and host-secreted mucus in a marine symbiosis and suggest conservation in the mechanisms of host-microbe interactions throughout the animal kingdom.