N-201. Citrate Synthase: A Functional Genetic Marker to Study Effects of Climate Drivers on Soil Microbial Communities

E. E. Austin, H. F. Castro, A. T. Classen, C. W. Schadt;
Oak Ridge Natl. Lab., Oak Ridge, TN.

Researchers need to better understand how climate change alters soil microbial community function if scientists are to make informed predictions on how climatic change may alter ecosystems. However, until recently few studies have used functional markers to asses the response of microbial soil communities to climatic stressors. Soil respiration is an integrator of soil microbial community function and is highly responsive to changes in temperature and moisture. Thus, citrate synthase was targeted as functional marker involved in this key metabolic process, to study how microbial communities may respond to climatic change. DNA were extracted from soils at the climatic multifactor site Old-field Community Climate and Atmospheric Manipulation at ORNL, where CO₂ (ambient, +300 ppm), warming (ambient, +3.5 ^oC) and soil moisture content (dry, wet) are manipulated in a constructed oldfield ecosystem. Primers targeting bacterial citrate synthase (gtlA) were designed based in previously deposited sequences in NCBI-GenBank and cloned libraries of the PCR-amplified citrate synthase gene were constructed; sequenced and phylogenetic analyses were conducted. The primers targeted a 225 bp fragment to be used afterward in quantification assays using real time PCR (QRT-PCR). The citrate synthase cloned libraries exhibited a great degree of diversity with all the major bacterial groups present in the neighbor-joining analysis. QRT-PCR conditions were optimized (annealing temperature 61^oC) and calibration curves highly linear (R²> 0.99) over six orders of magnitude (3.8x10⁵ to 3.8x10¹¹ copy number), and high amplification efficiencies (1.8 to 1.9) were achieved. Non-significant differences were observed among the climate treatments using citrate synthase QRT-PCR. However, comparison of microbial communities based citrate synthase using the phylogenetic comparison tool UniFrac revealed suggestive differences among the different climate treatments (P values of 0.06). Therefore, the microbial community composition maybe more responsive to climate drivers than the overall abundance of the functional marker.