Trace metal sample handling and analysis have led to the discovery that tiny (nanomolar) levels of metals such as iron are having profound influences on the diversity, abundance, and carbon fixation of primary producers in the oceans. One of these important primary producers, marine cyanobacteria, have similarly been shown to be affected, positively and negatively, by copper (natural and anthropogenic) levels and may also be influenced by cobalt and nickel levels. At the same time, cyanobacteria and other phytoplankton are changing the distributions of metals and their reactivity through uptake, which causes measurable depletion of metals in surface waters, and through the extra-cellular production of metal binding ligands. Because of the importance of copper in aquatic environments, the PIs will characterize copper metabolism in marine cyanobacteria using model Synechococcus strains from oligotrophic environments and from coastal environments that have very different metal physiologies revealed by the whole genome sequencing projects. They will characterize how the strains respond to different copper levels using whole genome microarrays that probe the global response of the cell and will combine these studies with state of the art characterization of intracellular metal levels and other measures of cellular physiology and photosynthetic capacity. Molecular genetics studies of diverse copper associated genes will be undertaken to determine their function in the cell. Preliminary results have found a potential candidate for an intracellular copper binding protein that is conserved in all marine cyanobacteria. At the same time, preliminary results have found that coastal cyanobacteria have greater resistance to copper than open ocean species and this might be due to a novel copper binding or efflux system. The PIs will illustrate the importance of copper in aquatic environments by developing hands-on lab components that will communicate some basic concepts around metal nutrition/pollution in the marine environment to middle school students. This will be undertaken in collaboration with Aquatic Adventures, who provide educational programs that connect underserved youth to science. Broader Impacts This research will reveal some of the major mechanisms by which marine cyanobacteria have adapted to metal levels in coastal and oligotrophic environments. Thus these results will help us understand the distribution and diversity of these organisms in relation to global primary productivity. They should lead to more robust biomarkers for metal stress and pollution in coastal environments. The PIs will expand on a previous outreach activity like their museum exhibit on marine genomics at the Birch Aquarium, La Jolla CA, by developing hands-on lab components that will communicate some basic concepts around metal nutrition/pollution in the marine environment to middle school students. This will be undertaken in collaboration with Aquatic Adventures, who provide educational programs that connect underserved youth to science, inspire environmental action, and increase exposure to marine habitats. In addition an undergraduate and graduate student in the interdisciplinary field of the metal physiology of microbes will be trained.
Copper is a major pollutant in coastal waters, and can also be introduced to pristine oligotrophic waters through atmospheric dust. Our goal was to understand copper toxicity to marine cyanobacteria of the genus Synechococcus, a major group of photosynthetic organisms at the base of aquatic food webs. We took advantage of available genomes for these cyanobacteria, functional genomics tools such as microarrays and proteomics, and the availability of genetic tools to modify specific genes in order to study their function. Intellectual Merit We carried out growth assays on a range of Synechococcus strains representing different "species" to exam and compare copper sensitivity. We found that coastal strains of marine Synechococcus were largely more tolerant to copper shock than open ocean strains. Thus copper appears to be a factor in the evolutionary speciation of this group. Synechococcus strains showed an osmoregulatory-like gene expression response, perhaps as a result of increasing membrane permeability. This could have implications for marine carbon cycling if copper stress in aquatic environment leads to dissolved organic carbon leakage in Synechococcus, thus increasing the growth rate of heterotrophic bacteria. We found that different Synechococcus strains also differed markedly in their response to copper. The open ocean strain (clade III) showed a general stress transcriptional response whereas the coastal strain (clade I) exhibited a more specifically oxidative acclimation response, that may be conferring copper tolerance. Using gene knockouts we found that the coastal strain used genes acquired by horizontal gene transfer to provide copper resistance. This is the first inactivation of any marine cyanobacterial "genomic island" gene, and is an example of how these islands of horizontally transferred genes may help strains adapt to environmental stress. The Synechoccocus population containing one copper resistance gene was estimated to be up to 10% of the population and show a distinct seasonal cycle in local waters. In general these and our other findings demonstrated specific mechanisms, both unique and conserved, by which Synechococcus responds to excess copper in their environment. These mechanisms were very different from those described in other model systems such as E. coli and thus highlight the importance of ecologically relevant model systems for studying environmental processes. Broader impacts We developed a classroom experiment for 5th graders to demonstrate the effects of copper pollution on phytoplankton by observing the damaging effects on phytoplankton swimming. Using NSF funds, projecting microscopes were purchased for Aquatic Adventures (later Ocean Discovery Institute--ODI) an organization bringing science to classrooms in under-served communities. After developing the curriculum with ODI, we later provided cells each year for this educational project. In 2013 the project was carried out with about 900 middle school students. This grant also trained one graduate student who recently completed her PhD and three undergraduates. Our results were communicated at scientific meetings and in the scientific literature. One of our major findings, that horizontally acquired genes can help cyanobacteria cope with a common environmental stress, suggests that as we study environmental change in the future, this mechanism may be very important and allow some organisms to adapt much faster to environmental change than might be expected through other evolutionary processes.
