1.  Biocomplexity of Aquatic Microbial systems:  Relating diversity of Microorganisms to Ecosystem Function
Agency:  NSF
Collaborators:  M. A. Voytek, USGS; G. A. Jackson, TAMU; P. Glibert, HPL; J. Collier, RPI; J. Zehr, UCSC

Microbial biogeochemical cycling of the elements regulates a dynamic environment in which the cycles of different elements are linked through the physiology of microorganisms.  While a certain degree of understanding can be gained through physical/chemical approaches to measurement and modeling of the net transformations, these approaches necessarily rely on gross simplifications about the role and regulation of the various functional groups (guilds) involved.  Recent advances in molecular microbial ecology have shown the microbial world to contain immense diversity and complexity at every level: redundancy and duplication of functional genes within a single organism; molecular diversity among functional genes that encode the same process in different organisms; large genetic diversity among different organisms apparently engaged in the same biogeochemical function within single communities; great variability in the species composition of different communities that apparently perform equally well.

The goal of this project is to investigate the functional relationship between complexity in microbial communities and the physical/chemical environment at a range of biological and ecological scales.  Previously, such analysis was technologically limited by the inability to assay large numbers of samples simultaneously for a large number of genes and phylotypes. Using gene array technology, we will be able to detect the distribution and differential expression of functional genes in natural systems. The results of this study will constitute the first step towards application of DNA chip technology for gene expression of "exotic" (i.e., not of biomedical importance) processes and organisms in the environment.  The gene arrays, along with a full suite of ecosystem process measurements, will be deployed along a transect that spans the eutrophic - oligotrophic gradient from the inland waters of the Chesapeake Bay out to the Sargasso Sea.  Experiments and functional gene studies will focus on key transformations in the carbon and nitrogen cycles (C fixation, N fixation, nitrification, denitrification, urea assimilation). The diversity of guilds will be interpreted in terms of ecosystem function, assessed using geochemical data and tracer experiments.  In addition to field studies designed to investigate and dissect the natural system, we will also perform perturbation experiments using mesocosms. The goal of these experiments is to determine how microbial species diversity affects the major energy and nutrient  flows within ecosystems, and to assess the degree of stability or instability associated with changes in redundancy within guilds of microorganisms responsible for major nitrogen and carbon pathways.
 

2.  Control of Denitrification in a Permanently Ice Covered Antarctic Lake: Potential for Regulation by Bioactive Metals
Agency:  NSF
CoPI:  M. L. Wells, University of Maine

Denitrification is the main loss term for fixed nitrogen from ecosystems, and thus its rate and regulation may directly affect primary production and carbon cycling over short and long time scales.  We propose to investigate the role of bioactive metals in regulating denitrification by exploiting a natural experimental system that occurs in a permanently ice-covered lake in the Taylor Valley of East Antarctica.  Chemical distributions in the two lobes of Lake Bonney imply that denitrification occurs in one lobe but not the other.  Most of the obvious biological and chemical variables that usually influence denitrification, and might account for the difference between the two lobes, have been ruled out by previous study.  Previous research has also demonstrated that denitrifying bacteria are present in both lobes of the lake and isolates derived from the lake show temperature and salinity optima consistent with their persistence in this environment.  We propose a combination of culture experiments and field work to resolve the mystery.

-- Growth experiments will determine the metal tolerances and metal requirements for growth and denitrification by the denitrifying isolates.

-- Total metal concentrations and metal speciation will be determined in surface transects and depth profiles, focusing mainly on the suboxic zone of both lobes of Lake Bonney.

-- These data sets will be combined to design  manipulative experiments in which the bioactive metal availability of lake water is altered in attempts i) to induce denitrification in incubations with water in which it appears to be inhibited and ii) to inhibit denitrification in water in which it appears to proceed "normally".

This approach will allow us to identify correlations between microbial activities and metal distributions in the field, and to test the roles of specific metals in both laboratory and simulated in situ experiments with organisms derived from the lake.  The relationships between metals and denitrification which we discover here are expected to shed light not only on Lake Bonney's unusual nitrogen cycle but, more generally, on the potential role of metals in regulation of microbial nitrogen transformations.  This insight will be very useful for evaluating the proposed use of paleo-denitrification indicators for past climate reconstructions, as well as the recent suggestion that small fluctuations in the global marine denitrification/nitrogen fixation ratio may have caused changes in atmospheric CO2 levels similar to those recorded over the last intergla-cial/glacial interval.
 

3.  The Coupling Between Carbon And Nitrogen Cycles In Coastal Upwelling  Ecosys-tems; Biogeochemical Cycling and its Molecular Basis
Agency:  DOE
CoPIs:  F. Wilkerson, SFSU; R. Dugdale, SFSU; J. Zehr, UCSC

This project establishes a research partnership between San Francisco State Uni-versity and the University of California at Santa Cruz and Princeton University with the aim of providing training opportunities in marine molecular biology for the diverse stu-dent body of SFSU. In addition to new research opportunities, we will develop a new SFSU lower division seminar-type undergraduate course entitled ãRecent Advances in Marine Scienceä to expose Biology and Geosciences undergraduates from diverse back-grounds to modern marine science.  This course would then be used as a vehicle, along with the Enrichment Office at SFSU, to reach out and recruit summer undergraduate in-terns supported by BIOMP scholarships to work in one of each of the collaborating insti-titions (SFSU, PU or UCSC laboratories).

Scientifically, this collaborative application proposes to study the response of the biological pump in ocean margins to nitrogen inputs by applying molecular techniques in concert with more traditional biogeochemical methods to trace the fate of upwelled carbon and nitrogen (primarily NO3) in an eastern boundary upwelling ecosystem.  The pro-posed research will use a primarily laboratory-based approach to develop probes and methods for applying them to field samples. The field samples will be collected along an upwelling plume off Bodega Bay, California, when winds are upwelling favorable during an on-going independently supported field program of cross shelf nitrogen and carbon coupling (NSF-CoOP, The Role of Wind-driven Transport in Shelf Productivity) which will provide the traditional biogeochemical framework to interpret the molecular studies.  Our hypothesis is that upwelling is a source of carbon and nitrogen which is then trans-formed by the biological pump, creating a carbon sink when new production by the eu-karyotic phytoplankton is maximal and the nitrogen and carbon assimilation are in bal-ance, followed by a period of NO3 depletion and regenerated productivity when  pro-karyotic primary production and carbon assimilation dominate. Our goal is to to evaluate the carbon and nitrogen links between new and total production of different microbial components with functional probes for the primary carbon (RuBP carboxylase, Ru-BisCO) and nitrogen assimilation (NO3 reductase, NaR and glutamine synthetase, GS) enzymes for a marine target prokaryote (Synechococcus) and eukaryote (a diatom, Skele-tonema costatum or Thalassiosira). The sequence information and probes are available for prokarytic RuBisCO, NaR and eukaryotic RuBisCO and GS but not for marine eukary-otic (diatom) NaR or Synechococcus GS. The diatom NaR gene has proved to be a chal-lenging gene to investigate in the past but we expect the three-way collaboration proposed using a variety of approaches should be successful.  Consequently our objectives are 1) to complete development of a probes for eukaryotic (preferably diatom) NaR and Synechococcus GS and 2) optimize protocols for nucleic acid extraction from seawater samples, and probe hybridization and 3) apply the C and N assimilation probes for dif-ferent taxonomic components of the microbial community to field samples from a coastal upwelling plume that have also been analyzed with biogeochemical tracers of carbon and nitrogen. This will enable us to detect which microbial carbon and nitrogen assimilation enzymes are expressed and functional in freshly and aged upwelled water, to evaluate when and where carbon and nitrogen cycling are weakly or tightly linked. Because the im-pact of upwelled nitrogen will be amplified through its coupling with the carbon cycle, knowing the time and space scales of eukaryotic and prokaryotic coupling of nitrogen and carbon cycles could be used to predict carbon burial and the system response to global change.
 

4.  Center for Environmental Bioinorganic Chemistry (CEBIC; Francois Morel, PI)
Agency:  DOE/NSF
Ward Section:   Denitrification

Trace metal limitation of denitrification :
We have investigated the trace metal limitations and requirements (Mo, Cd, Fe, Cu) for denitrifying bacteria by quantifying denitrification under trace-metal clean anoxic conditions, using trilaminate incubation bags. Several denitrifying strains were found to be unusually resistant to Cd toxicity. Both Cu and Fe could limit growth and denitrification by denitrifiers, and Cu in particular led to a terminal accumulation of N2O (Granger and Ward, in preparation).  Nitrous oxide reductase is a Cu enzyme and Cu limitation appears to act directly at this point in the pathway.  We hypothesize that Cu limitation may be involved in controlling the distribution of N2O in the ocean; regions where N2O accumulates(oxygen minimum zones) are also regions of very low Cu availability.  These observations are unusual and significant in that copper is more likely to be toxic than limiting for most microoganisms, and because it may be an example of control of biogeochemical cycles by metals other than iron.  Fieldwork in the Arabian Sea is planned for this fall with experiments designed to detect in situ Cu limitation.

Nitrite reductase
Our research on the biochemistry and molecular biology of denitrification focuses on nitrite reductase, the enzyme that catalyzes the reduction of nitrite to nitric oxide (NO).  This is a central enzyme in the pathway, often referred to as the denitrifying enzyme, because it converts fixed N to gaseous N, which is no longer available to most organisms.  The two forms of dissimilatory (respiratory) nitrite reductase, the cd-NiR (nirS gene) and Cu-NiR (nirK gene), are distributed across the Bacterial and Archaeal domains in a great diversity of microorganisms.

a) Diversity:
We have retrieved nirK and nirS genes from a large number and variety of microorganisms obtained from marine and sediment environments, from soils and halobenzoate degrading enrichments, and from uncultivated organisms using direct cloning (Song and Ward, submitted).  Phylogenetic analysis of the nir genes showed that with the well known exception of Pseudo-monas, both forms of nir are not found within a single genus.  The phylogeny of the nir genes is not congruent with the 16S rRNA phylogeny of the organisms, indicating widespread horizontal gene transfer of the components of denitrification.  As part of the planned Arabian Sea cruise, we will determine the relative abundance and activity of nirK and nirS genes in the denitrifying assemblage of the oxygen minimum zone.

b) Nitrifying bacteria
Both NiRs are well known in denitrifying bacteria, and the nitric oxide reductase  (NoR, which produces N2O) in denitrifiers has also been characterized.  It is also well known that nitrifying bacteria, obligate aerobic autotrophs, also produce both NO and N2O under some conditions, but the enzymology and genetics of these transformations in nitrifiers was not understood.  We discovered a nitrite reductase in nitrifying bacteria that is homologous with the nirK of denitrifiers.  NirK is present in several marine ammonia oxidizers which are closely related to Nitrosomonas marina, as shown in the phylogenetic tree below (nitrifier sequences in bold; Casciotti and Ward, 2001).  Similarly, we also recently discovered a norB gene in nitrifiers which is highly homologous with the norB gene from denitrifiers (Casciotti and Ward, in preparation).

We further investigated the similarities in the genes between denitrifiers and nitrifiers by determining the isotopic effects of the enzymes in whole cell and lysate assays.  For the residual nitrite, the isotopic effect is determined by the kind of NiR present, but is not influenced by whether the process is performed by a nitrifier or a denitrifier.  The isotopic effect must result from the mode of action of the iron vs. the copper enzyme, and perhaps the degree of exchange that occurs between the cell and the environment. The implication is that the significance of isotopic signatures of inorganic and gaseous nitrogen species in the ocean may be more complicated than previous suspected, if the effects of nitrification and denitrification cannot be distinguished.   A model is being developed to help sort out the expected isotopic fractionation in different environmental scenarios (Casciotti et al., in preparation).
 

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