Intellectual Merit: Pelagic marine bacteria and Archaea ("bacteria") play major roles in the variability in the biogeochemical fate of carbon fixed by phytoplankton in the ocean. The coupling of bacteria with the primary producers therefore has implications for climate and ecosystem models. Since bacteria interact with organic matter, including phytoplankton cells, at the nanometer to micrometer scale, understanding the biogeochemical coupling of bacteria with primary producers requires knowledge of the nature and strength of interactions at the micrometer scale. Using Atomic Force Microscopy (AFM) the PI recently discovered that a substantial fraction of heterotrophic (non-photosynthetic) bacteria, including the natural assemblages of Synechococcus, previously considered free-living, appeared conjoint with other bacteria. In a preliminary experiment, pelagic bacteria also became associated with cultures of Prochlorococcus (we do not yet know whether natural assemblages of Prochlorococcus harbor conjoint bacteria). In view of the biogeochemical importance of Synechococcus and Prochlorococcus as major primary producers in the ocean, their symbioses with heterotrophic bacteria could have far-reaching consequences.
This EAGER study has the potential to change ideas on microbial carbon cycling and marine ecosystems functioning. It may reveal a novel microspatial context, e.g., carbon and nutrient cycling 'hot spots', particularly in oligotrophic waters where Synechococcus and Prochlorococcus are major primary producers. This potentially transformative, but high risk research, is an excellent fit to the EAGER model for funding. Cutting edge methodologies, including cryo-electron tomography, nanoSIMS and single cell phylogenetics will be used through multidisciplinary collaborations to test hypotheses on the ultrastructure and ecosystem function of the symbioses. The phylogenetic identity of the conjoint partners will be determined by direct micromanipulation of seawater samples to pick individual conjoint cells followed by analysis by MDA (Multiple Displacement Amplification). NanoSIMS analysis of intercellular elemental exchanges will characterize the nature and biogeochemical significance of the symbioses. Field studies will examine the distribution of the symbioses in relation to relevant environmental factors. Success in the goals detailed by the PI should yield mechanistic understanding and biogeochemical significance of the symbioses. We will rapidly communicate our findings in a wide circulation journal as well as through talks at appropriate conferences.
Broader Impacts: This research project is aimed at gaining insights on fundamental mechanisms underlying the roles of microbes in the functioning of marine ecosystems, biological carbon cycling and the ocean's role in global climate variability. Therefore, the result should also be relevant to pressing societal issues fisheries and climate change. The PI's lab is actively involved in public education from local to international levels, and the findings of the proposed research will be disseminated broadly to the public in appropriately accessible formats. One postdoctoral fellow and one graduate student will receive training during this research.
In the ocean, microorganisms are critical in maintaining ecosystem functioning and the grand cycles of carbon and other elements that affect ocean health and global climate. About one-half of global photosynthesis occurs in the ocean, resulting in massive CO2 –carbon incorporation into organic matter as well as substantial production of oxygen that we breathe. A major fraction of this large-scale marine photosynthesis is due to abundant cyanobacteria that occur in the upper ocean. As the ocean’s carbon cycle undergoes change bacteria (including Archaea) decompose much of the organic matter synthesized by photosynthetic microbes. If the organic matter is decomposed within the upper ocean then the CO2 produced can exchange readily with the atmosphere; the CO2 released from organic matter that sinks deeper before being decomposed stays at depth for centuries. Therefore, the strength of coupling between organic matter and heterotrophic bacteria (that use organic matter for energy and growth) is critical for understanding the global carbon cycle and climate change. Phytoplankton-bacteria coupling is generally studied in the context of bacterial use of organic matter released from phytoplankton into seawater. Using nanometer resolution imaging with an Atomic Force Microscope (AFM) we discovered that cyanobacteria (Synechococcus spp.) occur attached to other bacteria. This raised the possibility of physical coupling between photosynthetic bacteria and heterotrophic bacteria. Their interaction raises question of the nature of their relationship—they could be involved in symbiosis (helping one another with needed nutrients); antagonism (one may be biochemically attacking the other); or neutral (e.g. incidental association, inconsequential to the fitness of the other). If the relationship entailed resource sharing/exchange then it might cause tight coupling for carbon flux and nutrient cycling—since released solutes could be used at high local concentration (and without competing with other bacteria in environment). Synechococcus could supply organic matter to bacteria and bacteria could supply inorganic nutrients to the Synechococcus. Our EAGER grant objective was to address questions on the nature of interactions between Synechococcus and bacteria: How widely distributed are the conjoint Synechococcus-heterotrophic in the ocean? What is the phylogenetic affiliation of the associated heterotrophic bacteria? Does the association confer a growth advantage or disadvantage to the interaction cells-or is there no effect on growth rates? Is there carbon and/or nitrogen exchange among the interacting microbes? Our main findings are: 1) Field observations. We examined seawater from North Pacific Gyre, Sargasso Sea, and Northern Adriatic Sea, and Southern California coastal waters. Cyanobacteria-heterotrophic bacteria association in all sets of samples the proportion of conjoint Synechococcus was quite variable, 10-80%. In Sargasso Sea samples the conjoint Synechococcus-heterotrophic bacteria accounted was low, 3-16%; in samples from north Pacific had the highest % conjoint Synechococcus, up to 80%. The conclusion was that Synechococcus-heterotrophic bacteria associations occur broadly, both in oligotrophic and mesotrophic waters. 2) Phylogenetic specificity of conjoint partners. We picked individual conjoint pairs by micromanipulation and did genome amplification. We identified two associated bacterial taxa related to an Alpha-Proteobacterium (100% identity to Pelagibacter ubique 16S rRNA gene sequence) and to a Gamma-Proteobacterium (99% identity to a 16S rRNA sequence found during a phytoplankton bloom; NCBI accession number EU799583). 3) Carbon and nitrogen exchange between bacteria and Synechococcus. We used two individual-cell techniques (14C-microautoradiography and 13C and 15N-NanoSIMS). Their appears to be a growth advantage for Synechococcus to be associated with heterotrophic bacteria. However, data suggest some associations may involve antagonism. However, more analysis (underway) is needed to clarify this question. 4) Laboratory model system for conjoint cells and their utility for interaction studies. We developed a model systems of two cultured Synechococcus strains with bacteria (Vibrio, Pseudoalteromonas, Alteromonas, Flavobacterium). We used confocal video microscopy to study the spatial and behavioral interactions between Synechococcus and heterotrophic bacteria. The bacteria were attracted towards Synechococcus cells, displaying a range of behavioral patterns include frequent close encounters. This model system will enables future studies on interactions. 5) Molecular bases of recognition and interaction: We examined how the organic matter concentration affects the association. Nutrient enrichments reduced % conjoint cells. We also discovered a role for specific cell surface proteins (lectins) in recognition and mediating the associations. 6) Development of individual cell growth rate method: We developed a sensitive method to measure individual cell growth rate. We found that 42% of the conjoint heterotrophic bacteria were growth-positive whereas 98% of the free heterotrophic bacteria were growth-positive. This result supports the possibility that some associations involve antagonistic interactions. 7) NanoSIMS analysis on elemental fluxes among conjoint bacteria. We performed stable isotope carbon and nitrogen labeling experiments on the natural microbial populations to test whether there was exchange of newly fixed carbon and nitrogen from cyanobacteria to the associated bacteria. We did not find significant differences for carbon and nitrogen exchange in the free- versus cyanobacteria-associated heterotrophic bacteria.
