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2011 Annual Science Report

Arizona State University Reporting  |  SEP 2010 – AUG 2011

Stoichiometry of Life - Task 1 - Laboratory Studies in Biological Stoichiometry

Project Summary

This project component involves a diverse set of studies of various microorganisms with which we are trying to better understand how living things use chemical elements (nitrogen, phosphorus, iron, etc) and how they cope, in a physiological sense, with shortages of such elements. For example, how does the “elemental recipe of life” change when an organism is starved for phosphorus? Is this change similar for diverse species of microorganisms? Are the changes the same if the organism is limited by a different key nutrient? Furthermore, how does an organism shift its patterns of gene expression when it is starved by various nutrients? This will help in interpreting studies of gene expression in natural environments. At an even more profound level: can an organism substitute an element that is similar to the one that is limiting, as in the case of arsenic for phosphorus?

4 Institutions
3 Teams
6 Publications
0 Field Sites
Field Sites

Project Progress

Task 1a: Extended Redfield Ratio under nutrient limitation.
In late summer 2011 visiting PhD student Albert Rivas-Ubach (Autonomous University of Barcelona) began work with Synechocystis sp. PCC 6803 in our laboratory chemostats under P-, N-, and Fe-limitation. As this report is being prepared, the chemostats are reaching equilibrium and biomass will be harvested for analysis of elemental and biochemical composition along with gene expression. Rivas-Ubach is an expert on metabolomics via mass spectrometry and so these samples will also be processed for their metabolic profiles. Once completed, similar studies will begin with the mesophilic bacterium Bacillus sp. m3-13 from Cuatro Cienegas, Mexico.

Task 1b: Interactions of molybdenum with other biogeochemical cycles.
I. Mo storage protein Mop in cyanobacteria: In order to determine the regulation of the putative Mo storage protein “Mop” in freshwater heterocystous cyanobacteria, Glass and undergraduate research assistant Eric Hughes performed various experiments manipulating Mo supplies to with the model organism Nostoc sp. PCC 7120. Complex dynamics during acclimation and transition to Mo limitation suggest a complex regulation of Mo storage by this heterocystous cyanobacterium, including transcriptional co-regulation of mop and nif and post-translational changes in Mop protein subunit composition as a function of cellular Mo. These results were presented at the 2010 International BioMetals Symposium, the 2010 Environmental Bioinorganic Chemistry Gordon conference, the 2011 Geobiology Gordon conference and the 2011 AbGradCon. This research comprised a chapter in Glass’ PhD dissertation (successfully defended in March 2011) will be submitted to the journal Applied and Environmental Microbiology in October or November 2011. Furthermore, undergraduate researcher Zureyma Martinez evaluated the distribution of Mop genes at Yellowstone National Park, processing samples taken in summer 2010. This work was presented at the April 2011 Southern California Geobiology Symposium at University of Southern California.

II. Mo-nitrate co-limitation at Castle Lake: During 2009 and 2009 Glass performed various field experiments testing Mo-nitrate interactions at N- (and Mo?) deficient-Castle Lake, California. The data study lends further support to the hypothesis that low Mo can limit nitrate assimilation in freshwaters with typical low Mo levels (<5 nM) when ammonium is scarce, possibly resulting in decreased capacity of terrestrial ecosystems to serve as sinks for anthropogenic carbon dioxide emissions. These results were presented at the 2010 ASLO-NABS Summer Meeting and comprised a chapter in Glass’ PhD dissertation (successfully defended in March 2011). A paper on this research will be submitted to Aquatic Microbial Ecology in October or November 2011. Another paper on the geochemistry of Mo cycling in Castle Lake is in preparation for the journal Geochimica Cosmochimica Acta.

III. Nickel-molybdenum co-limitation and evolution of Mo-nitrogenase: Glass developed and presented a hypothesis in collaboration with Eric Boyd, Steve Romaniello and Ariel Anbar connecting ocean redox chemistry, dynamic Ni / Mo / V / Fe availabilities, and nitrogenase / hydrogenase interactions and evolution, ultimately arguing that these early events in biogeochemical evolution were responsible for the widespread adoption of Mo-based nitrogenases in modern biogeochemistry. This hypothesis was presented at the 2011 NAI Workshop Without Walls on Molecular Paleontology. It was also used to develop a mock proposal at the 2011 Astrobiology Research Focus Group workshop. Experiments testing components of hypothesis are needed before publication can be pursued.

Task 1c: Arsenic substitution
Wolfe-Simon and colleagues in Oremland’s lab at the USGS (Menlo Park) published data in Science supporting the hypothesis that arsenic can substitute for phosphorus in biomolecules – a hypothesis published previously by Wolfe-Simon, Anbar and Davies.

More specifically, Wolfe-Simon et al. successfully cultivated 14 aerobic isolates of aerobic microbes, from sediments and waters collected from Mono Lake, CA, that persisted when cultured in artificial medium (basal salts) supplemented with 10 mM glucose, a full compliment of vitamins and trace metals and 5 mM arsenate (AsO43-) with no added phosphate (PO43-). One isolate, strain D (later identified as “GFAJ-1”) proved most robust, with a relative fast growth rate compared to other aerobic isolates. This strain was used for initial detailed study. Growth curves demonstrated growth of GFAJ-1 when given PO43- (​As/+P) but also when given AsO43and no PO43- (+As/-P) over the control condition given neither PO43- nor AsO43- (-As/-P). To determine the intracellular distribution of elemental arsenic, the biomass was subjected both ICPMS and NanoSIMS analyses. Both methods indicated that arsenic was contained within the cells. In radiolabelling studies, 73AsO43- was shown distributed into nucleic acid, protein, small molecular weight metabolite and lipid cellular fractions in cells grown with the radiolabel as 73AsO43-. Additional evidence was gathered via three types of x-ray spectroscopy to further describe and characterize the intracellular arsenate. XRF confirmed intracellular arsenic while mXANES and mEXAFS showed the intracellular arsenic was as arsenate, As(V), in an environment chemically consistent with phosphate. That is, arsenic bound to about four oxygen atoms with distal carbons. This structural information supported the distribution and basic biochemical environment of arsenic as As(V) in a similar configuration to phosphate in a DNA backbone or potentially other biomolecules like small molecular weight metabolites. A gel band of extracted DNA that had been run on an agarose gel was also analyzed by NanoSIMS. It showed arsenic associated with the genomic DNA band.

The Science paper inspired widespread discussion and commentary in the scientific community as well as the general public. To that end, critical commentaries were published in Science and answered via a “Technical Response to Comments”.