Phylogenetic diversity of bacterioplankton in relation to distribution of cell-specific physiological properties and environmental conditions in an upwelling ecosystem

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Marine bacterioplankton are central and quantitatively significant components of marine ecosystems; their role looms large in determining the capacity of oceanic systems to sequester atmospheric CO2 into the deep ocean. Current models constructed to examine these questions consider marine bacterioplankton as a monolithically active compartment. This is in large part due to the lack of understanding regarding whether specific phylogenetic or physiologically identifiable groups of bacteria have distinct conditions for optimal growth, or whether diverse groups of bacterioplankton do in fact respond uniformly to changing environmental conditions, as the models assume. Flow cytometric enumeration of marine bacterioplankton using nucleic-acid specific stains has shown the routine presence of two distinct clusters of bacterial cells based on cell-specific nucleic acid (NA, i.e. DNA and RNA) content. Opposing ideas have been proposed concerning these distinct clusters of bacteria: 1) most bacterial activity is due to cells with higher nucleic acid content (high-NA), while lower nucleic acid content cells (low-NA) are either slow-growing or dead, or 2) both high-NA and low-NA cells have similar biomass-specific rates of activity. This research will focus on the high-NA and low-NA cells present in bacterial communities in the Oregon upwelling ecosystem. Two alternative hypotheses will be examined: Ho 1. High-NA and low-NA bacterial cells are composed of phylogenetically distinct bacterial assemblages that show differences in physiological activity. This would be the case if high NA and low-NA cells tend to fall into separate assemblages of cells that are adapted to different growth strategies. Ho 2- High-NA and low-NA cells tend to have similar phylogenetic compositions and similar biomass-specific rates of activity. The approach will involve flow cytometric enumeration and sorting of bacteria based on cell-specific nucleic acid or protein content. Sorted bacteria will be assayed for cell-specific activity via prior labeling with radioactive substrates, and assayed for phylogenetic diversity via PCR amplification of DNA extracted from sorted cells followed by DGGE analysis, sequencing of selected gel bands, and phylogenetic identification. At the same time, radiolabeled subsamples will be collected on filters and analyzed by combined microautoradiography and fluorescence in situ hybridization (FISH) using domain-specific probes, as well as more specific probes, in order to determine the relative contribution of substrate-active phylogenetic groups to total bacterial abundance. A main goal of the project is to put the results in ecological context, i.e. to relate variability in activity and diversity among high-NA and low-NA bacterioplankton to environmental parameters likely to affect the growth of marine bacteria (e.g. potential bottom-up controls such as phytoplankton biomass and riverine DOC influx). Intellectual merit: This research project will address the linkages between physiological and phylogenetic diversity in marine bacterioplankton, and how such linkages might affect bacterial processing of organic matter in marine systems. The results would be a significant step in understanding the role of specific phylogenetic components of the bacterioplankton community in global biogeochemical cycles and carbon flux. Broader impacts: This project will support the Ph.D. thesis research of a female graduate student and provide the student with post-doctoral training; it will also enhance the teaching efforts of the P.I..s and support the common use Flow Cytometry Facility and molecular genetics capabilities in the College of Oceanography and Atmospheric Sciences.