Astrobiology Science and Technology for Exploring Planets (ASTEP)

PI: Newman, Dianne

The advent of oxygenic photosynthesis was a pivotal event in the formation of the modern biosphere and the evolution of complex life. However, our constraints on the timing of this event are presently very rough. The occurrence of molecular fossils known as 2-methylhopanes in the rock record has been taken as evidence that photosynthetically-derived O ₂ first appeared on Earth at least 2.7 billion years ago. This assumption arises from the fact that modern cyanobacteria, organisms that engage in oxygenic photosynthesis, produce significant quantities of 2- methylbacteriohopanepolyols (2MeBHPs), progenitors of 2-methylhopanes. Nevertheless, a number of independent proxies indicate that a major global redox transition occurred roughly 400 million years later (2.3 Ga). If cyanobacteria were present and engaging in oxygenic photosynthesis at 2.7 Ga, why did it take so long for the Earth to become oxygenated? Is the assumption that 2-methylhopanes are biomarkers for oxygenic photosynthesis correct, or might they reflect some other property of cyanobacteria that is decoupled from their capacity to generate O ₂ ? To date, no evidence exits that 2MeBHPs and oxygenic photosynthesis are functionally related. Based on recent evidence, we hypothesize that the presence of 2-MeBHPs may be related to the localization of a carbon-concentrating mechanism (CCM) in the membranes of cyanobacteria, rather than to oxygen production. If correct, this hypothesis implies that 2- methylhopanes record the evolution of cyanobacterial autotrophy rather than oxygenic photosynthesis per se. The specific aims outlined below will lay the groundwork for exploring the function of 2-MeBHPs in cyanobacteria.

1.) Determine where 2-MeBHPs localize within the cell. Cyanobacteria possess a relatively complex membrane arrangement, composed of an outer membrane (OM), a plasma membrane (CM), and a photosynthetically-active thylakoid membrane system ™ derived from the CM. We will isolate these subcellular fractions and quantitatively analyze the distribution pattern of BHPs and 2-MeBHPs within two different cyanobacteria: Phormidium luridum and Synechocystis PCC 6803.
2.) Characterize the conditions under which 2-MeBHPs are produced. Through quantitative analysis of the production of 2-MeBHPs and measurement of carbon uptake rates by P. luridum and PCC 6803, we will test the hypothesis that conditions that affect the CCM also affect 2-MeBHP production. 2-MeBHP production will be measured under conditions where pCO ₂ , salinity and pH are independently varied. In addition, we will examine the relationship between 2-MeBHP production and mode of metabolism (photoautotrophy v. (photo)heterotrophy), as well as its relationship to growth rate and temperature.
3.) Identify the gene (or set of genes) that encodes the enzyme that methylates the C-2 atom of 2-MeBHPs. Standard techniques in bacterial genetics will be utilized to determine the gene(s) that are responsible for C-2 methylation of BHPs. One of two approaches will be used, depending on the results of the localization and regulation experiments: (i) targeted mutations in PCC 6803 will be made based on microarray analyses, (ii) DNA from P. luridum will be expressed in PCC 6803 and the methylase gene will be identified by identifying the smallest gene fragment that confers the ability to make 2-MeBHPs. Upon identification of the C-2 methyltransferase, we will compare its sequence to others in the database to explore its phylogenetic relationship to other gene products as well as its distribution amongst sequenced microorganisms.

This collaborative effort will reveal novel aspects of biomarker biosynthesis and function, and, thus, promises to improve our ability to interpret the evolution of metabolism on Earth. This fulfills the stated goal of the Early Evolution of Life and the Biosphere component of the solicitation announcement NNH05ZDA001N-EXB (Astrobiology: Exobiology & Evolutionary Biology).