Elena Kazamia

Mutualistic interactions between vitamin B12‐dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B(12) (cobalamin), which they need as a cofactor for B(12) -dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B(12) -dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B(12) -dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi-continuous culture, indicating a degree of regulation. However, addition of either vitamin B(12) or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B(12) for growth because it encodes a B(12) -independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B(12) . Co-culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B(12) -independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.

Direct exchange of vitamin B12 is demonstrated by modelling the growth dynamics of algal–bacterial cocultures

The growth dynamics of populations of interacting species in the aquatic environment is of great importance, both for understanding natural ecosystems and in efforts to cultivate these organisms for industrial purposes. Here we consider a simple two-species system wherein the bacterium Mesorhizobium loti supplies vitamin B12 (cobalamin) to the freshwater green alga Lobomonas rostrata, which requires this organic micronutrient for growth. In return, the bacterium receives photosynthate from the alga. Mathematical models are developed that describe minimally the interdependence between the two organisms, and that fit the experimental observations of the consortium. These models enable us to distinguish between different mechanisms of nutrient exchange between the organisms, and provide strong evidence that, rather than undergoing simple lysis and release of nutrients into the medium, M. loti regulates the levels of cobalamin it produces, resulting in a true mutualism with L. rostrata. Over half of all microalgae are dependent on an exogenous source of cobalamin for growth, and this vitamin is synthesised only by bacteria; it is very likely that similar symbiotic interactions underpin algal productivity more generally.

High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform

Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.

Fundamental shift in vitamin B12 eco-physiology of a model alga demonstrated by experimental evolution.

A widespread and complex distribution of vitamin requirements exists over the entire tree of life, with many species having evolved vitamin dependence, both within and between different lineages. Vitamin availability has been proposed to drive selection for vitamin dependence, in a process that links an organism’s metabolism to the environment, but this has never been demonstrated directly. Moreover, understanding the physiological processes and evolutionary dynamics that influence metabolic demand for these important micronutrients has significant implications in terms of nutrient acquisition and, in microbial organisms, can affect community composition and metabolic exchange between coexisting species. Here we investigate the origins of vitamin dependence, using an experimental evolution approach with the vitamin B12-independent model green alga Chlamydomonas reinhardtii. In fewer than 500 generations of growth in the presence of vitamin B12, we observe the evolution of a B12-dependent clone that rapidly displaces its ancestor. Genetic characterization of this line reveals a type-II Gulliver-related transposable element integrated into the B12-independent methionine synthase gene (METE), knocking out gene function and fundamentally altering the physiology of the alga.

Assessing the environmental sustainability of biofuels

Biofuels vary in their potential to reduce greenhouse gas emissions when displacing fossil fuels. Savings depend primarily on the crop used for biofuel production, and on the effect that expanding its cultivation has on land use. Evidence-based policies should be used to ensure that maximal sustainability benefits result from the development of biofuels.

How mutualisms arise in phytoplankton communities: building eco-evolutionary principles for aquatic microbes.

Abstract Extensive sampling and metagenomics analyses of plankton communities across all aquatic environments are beginning to provide insights into the ecology of microbial communities. In particular, the importance of metabolic exchanges that provide a foundation for ecological interactions between microorganisms has emerged as a key factor in forging such communities. Here we show how both studies of environmental samples and physiological experimentation in the laboratory with defined microbial co‐cultures are being used to decipher the metabolic and molecular underpinnings of such exchanges. In addition, we explain how metabolic modelling may be used to conduct investigations in reverse, deducing novel molecular exchanges from analysis of large‐scale data sets, which can identify persistently co‐occurring species. Finally, we consider how knowledge of microbial community ecology can be built into evolutionary theories tailored to these species’ unique lifestyles. We propose a novel model for the evolution of metabolic auxotrophy in microorganisms that arises as a result of symbiosis, termed the Foraging‐to‐Farming hypothesis. The model has testable predictions, fits several known examples of mutualism in the aquatic world, and sheds light on how interactions, which cement dependencies within communities of microorganisms, might be initiated.

Cross-exchange of B-vitamins underpins a mutualistic interaction between Ostreococcus tauri and Dinoroseobacter shibae

Ostreococcus tauri, a picoeukaryotic alga that contributes significantly to primary production in oligotrophic waters, has a highly streamlined genome, lacking the genetic capacity to grow without the vitamins thiamine (B1) and cobalamin (B12). Here we demonstrate that the B12 and B1 auxotrophy of O. tauri can be alleviated by co-culturing with a heterotrophic bacterial partner Dinoroseobacter shibae, a member of the Rhodobacteraceae family of alpha-proteobacteria, genera of which are frequently found associated with marine algae. D. shibae lacks the complete pathway to synthesise three other B-vitamins: niacin (B3), biotin (B7), and p-aminobenzoic acid (a precursor for folate, B9), and the alga is in turn able to satisfy the reciprocal vitamin requirements of its bacterial partner in a stable long-term co-culture. Bioinformatics searches of 197 representative marine bacteria with sequenced genomes identified just nine species that had a similar combination of traits (ability to make vitamin B12, but missing one or more genes for niacin and biotin biosynthesis enzymes), all of which were from the Rhodobacteraceae. Further analysis of 70 species from this family revealed the majority encoded the B12 pathway, but only half were able to make niacin, and fewer than 13% biotin. These characteristics may have either contributed to or resulted from the tendency of members of this lineage to adopt lifestyles in close association with algae. This study provides a nuanced view of bacterial–phytoplankton interactions, emphasising the complexity of the sources, sinks and dynamic cycling between marine microbes of these important organic micronutrients.