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Project Progress

Task 1. Field Studies and Laboratory Characterization of Hydrothermal Vent Microbes (Baross).
We have completed the characterization of unique subseafloor hyperthermophiles. One of the strains was found to oxidize acetate anaerobicallly using Fe(III) as the electron acceptor. In the process the Fe(III) is converted to magnitite. This is the first hyperthemophilic archaea to show this physiology and the first unique subsurface hyperthermophile isolated. This organism is a new genus within the archaeal family Thermococcales and one of the deepest roots in the Archaea phylogenetic tree. A manuscript describing this organism is being prepared for publication in Science.

The molecular-phylogenetic analyses of the subsurface microbial communities from the 1998 deep-sea eruption at Axial Volcano, Juan de Fuca Ridge, has been completed for samples collected in 1998 and 1999. There is a huge diversity of both archaea and bacteria in these subsurface water samples, most of which are not related to any known cultured organisms. Some of the archaeal sequences form clusters that are not related to any reported environmental sequences. We are in the process of trying to decipher the physiological characteristics of these uncultured archaea using molecular methods and novel culturing procedures.

A unique thermophilic group of bacteria was isolated from Axial Volcano subsurface fluids. One of these strains grows in extremely low levels of organic material or with carbon dioxide and hydrogen. These organisms produce a significant amount of extracellular polysaccharides used in biofilm formation. These organisms are unrelated to any known cultured bacteria and could be a new family of thermophilic bacteria. A manuscript describing this subsurface diversity is being prepared. We also will return to Axial in July 2000 to obtain additional samples.

A molecular probe has been designed to detect the NIF (nitrogen fixation) gene in archaea and has been used successfully to detect the NIF gene in hyperthermophiles from subseafloor environments. We are currently in the process of cloning and expressing these archaeal NIF genes.

A preliminary description of the microbial ecology of active sulfide chimneys has been completed using a combination of analyses. These analyses include environmental scanning electron microscopy, EDAX, epifluorescence microscopy including Fluorescence In Situ Hybridization (FISH) to provide quantitative estimates of total cell numbers and the abundance of specific phylogenetic domains, and phylogenetic analyses of the microbial communities using 16S rRNA gene sequences. These data show that only archaea inhabit the high temperature zones of the sulfide. There is evidence that intact microbial colonies exist in mineral zones that are at temperatures greater than 150°C. A manuscript is being prepared for Science.

Task 2. Stable Isotope Studies of Hydrothermal Organisms (Emerson, Fogel).
We have started investigations into the stable isotopic compositions of hydrothermal vent organisms grown in culture at the American Type Culture Collection in Manassas, Virginia. Isotopic experiments were designed to test whether we could detect differences in central metabolic pathways through the analyses of carbon isotopic compositions of individual amino acids. For these experiments, a variety of Archae- and Eubacteria with different metabolic pathways were cultured and analyzed. In addition, equipment for culturing microorganisms was purchased and installed at the Geophysical Laboratory to provide supporting microbial material for isotopic analyses.

Isotopic compositions of amino acids can be used to determine branch points in metabolic synthesis of amino acids. In addition, we are comparing metabolic pathways in different organisms from various positions in the Universal Tree by investigating isotopic fractionation of particular amino acids relative to others. For example, glutamic acid and aspartic acid are both synthesized by the TCA cycle, or Krebs cycle. The TCA cycle operates either in the forward or the reverse direction in microbes, depending on the organism or the growth conditions. It is often assumed that the enzymes responsible for synthesis of amino acids are fairly conservative. We have found that we are able to distinguish different pathways and different enzymes with differential fractionation of carbon isotopes in amino acids. The long term goal of this research is to be able to link known physiology to a particular carbon isotopic fractionation.

In concert with this work, we have been investigating the stability of proteins in geochemical environments. With sensitive protein assays, we have determined that proteins from primary photosynthetic producers turn over rapidly and the original isotopic composition is almost completely scrambled. Experiments are beginning with new methodology to be able to recognize and assign chemical structures and molecular weight to complex diagenetic organic matter. To this end, we have used stable isotopes as tracers and are developing methodology for determining the D- and L-enantiomer ratios and isotopic ratios of geochemically-derived amino acids. Samples from crustal fluids will be analyzed for D- and L-amino acids and their corresponding isotopic compositions to provide an overall framework for abiotic or biological synthesis of organic matter in hydrothermal environments.