The ISME Journal (2007) 1, 163–179 & 2007 International Society for Microbial Ecology All rights reserved 1751-7362/07 $30.00 www.nature.com/ismej ORIGINAL ARTICLE Functional microarray analysis of nitrogen and carbon cycling genes across an Antarctic latitudinal transect Etienne Yergeau1, Sanghoon Kang2, Zhili He2, Jizhong Zhou2 and George A Kowalchuk1,3 1Center for Terrestrial Ecology, Netherlands Institute for Ecology (NIOO-KNAW), Heteren, The Netherlands; 2Department of Botany and Microbiology, Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA and 3Institute of Ecological Science, Free University of Amsterdam, Amsterdam, The Netherlands Soil-borne microbial communities were examined via a functional gene microarray approach across a southern polar latitudinal gradient to gain insight into the environmental factors steering soil N- and C-cycling in terrestrial Antarctic ecosystems. The abundance and diversity of functional gene families were studied for soil-borne microbial communities inhabiting a range of environments from 511S (cool temperate – Falkland Islands) to 721S (cold rock desert – Coal Nunatak). The recently designed functional gene array used contains 24 243 oligonucleotide probes and covers 410 000 genes in 4150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance and organic contaminant degradation (He et al. 2007). The detected N- and C-cycle genes were significantly different across different sampling locations and vegetation types. A number of significant trends were observed regarding the distribution of key gene families across the environments examined. For example, the relative detection of cellulose degradation genes was correlated with temperature, and microbial C-fixation genes were more present in plots principally lacking vegetation. With respect to the N-cycle, denitrification genes were linked to higher soil temperatures, and N2-fixation genes were linked to plots mainly vegetated by lichens. These microarray-based results were confirmed for a number of gene families using specific real-time PCR, enzymatic assays and process rate measurements. The results presented demonstrate the utility of an integrated functional gene microarray approach in detecting shifts in functional community properties in environmental samples and provide insight into the forces driving important processes of terrestrial Antarctic nutrient cycling. The ISME Journal (2007) 1, 163–179; doi:10.1038/ismej.2007.24; published online 24 May 2007 Subject Category: geomicrobiology and microbial contributions to geochemical cycles Keywords: functional gene microarray (GeoChip); antarctic soil ecosystems; microbial nitrogen cycle; microbial carbon cycle; real-time PCR Introduction the interrelated processes. Furthermore, some Antarctic ecosystems are being subjected to unpre- Biogeochemical cycles in Antarctic terrestrial habi- cedented human-induced climate change, the rami- tats are almost exclusively driven by microbial fications of which remain mostly unknown. activities. Indeed, due to a principal lack of insect Although the nitrogen cycle in terrestrial Antarctic and mammalian herbivores and detritivores at most is as yet poorly understood, available evidence locations (Smith and Steenkamp, 1992), food webs suggest that nitrogen is the main limiting factor in are primarily driven via bacterial and fungal high altitude and high latitude ecosystems (Mataloni detritus-based routes (Smith, 1994). Given their et al., 2000; Shaw and Harte, 2001; Solheim et al., relative trophic simplicity, such ecosystems repre- 2004). Thus, even small changes in the nitrogen sent useful models for linking primary production cycle, as predicted in climate change scenarios and microbial nutrient cycling and disentangling (Barnard et al., 2005), are hypothesized to induce disproportionately large shifts in the dynamics of such ecosystems. However, without any recent Correspondence: Professor GA Kowalchuk, Center for Terrestrial baseline study across a range of environments, the Ecology, Netherlands Institute for Ecology (NIOO-KNAW), PO effects of perturbations on Antarctic soils are hard to Box 40, 6666ZG, Heteren, The Netherlands. E-mail: [email protected] predict. In the Antarctic, it was reported that the Received 26 January 2007; revised 19 March 2007; accepted 20 main source of nitrogen in soils was either from March 2007; published online 24 May 2007 precipitation of volatilized ammonium when in Soil microbial N- and C-cycles in Antarctica E Yergeau et al 164 proximity of bird colonies (Christie, 1987) or from ogy have focused on determinations of community N2-fixation by cyanobacteria (Ino and Nakatsubo, structure based upon phylogenetic markers such as 1986). N2-fixation in Antarctica has also been 16S rRNA genes (Gentry et al., 2006). Although such suggested to occur via the action of free-living N2- approaches provide powerful and detailed pictures fixing bacteria and the cyanobionts of lichens (Line, of microbial community structure in complex 1992). However, heterotrophic N2-fixation was not environmental samples, they generally provide little reported as a significant source of reduced nitrogen insight into microbial functions. In recent years, (Pandey et al., 1992), and heterotrophic bacteria are major efforts have therefore been into the targeting believed to rarely fix N2 in these environments of other functional genes that might provide insight because of energy limitations (Christie, 1987). In the beyond pure phylogenetic characterizations. A few other studies addressing the N-cycle in Antarctic robust 50-mer functional gene array platform has habitats, Signy Island habitats were reported to be been recently demonstrated to be useful in examin- devoid of ammonia-oxidizing bacteria (Vishniac, ing multiple functional gene targets, mainly within 1993), and nitrate-respiring bacteria outnumbered key gene families involved in microbial nutrient denitrifying bacteria, suggesting N conservation in cycling and contaminant degradation (Rhee et al., some Antarctic soils (Christie, 1987). 2004; Tiquia et al., 2004). With the expansion of Available evidence suggests that fungi are the available databases and probe design improvements dominant decomposers in Antarctic ecosystems, (He et al., 2005; Li et al., 2005), this platform has representing a sharp contrast to Arctic systems, recently been expanded to allow for the simulta- where bacteria dominate this function (Walton, neous detection of over 10 000 gene variants on a 1985). Vegetation type is also thought to play an single array (He et al., 2007). important role in driving microbial decomposers. In The main goal of the present study was to describe South Georgia (maritime Antarctic), very marked the functional aspects of microbial communities differences in cellulose degradation were observed involved in soil-borne nutrient cycling across an under different vegetations, and it was concluded Antarctic latitudinal gradient. Toward this goal, we that Antarctic decomposition is linked to vegetation used a recently designed functional gene microarray type, substratum potential and relative dominance system, the GeoChip (He et al., 2007), containing of fungi (Walton, 1985). Freeze–thaw cycles are also probes for the known diversity of the most impor- believed to play an important role in the carbon tant microbial driven nutrient cycle processes, cycle in Antarctica, since they change exudation especially the N- and C-cycles. Functional commu- patterns in cryptogams and impose stress upon nity analyses focused on soil samples taken from resident microbes (Tearle, 1987; Melick and Seppelt, different soil environments at five sites with 1992; Melick et al., 1994). In addition to differences latitudes ranging from 511S (cool temperate – Falk- in litter quantity and quality, the major soluble land Islands) to 721S (cold rock desert – Coal carbohydrate exudates also differ between higher Nunatak) and included a comparison of vegetated plants, bryophytes and lichens (Melick and Seppelt, versus principally bare sites. We provide a detailed 1992; Melick et al., 1994), providing an additional analysis of functional community structure across mechanism by which vegetation type can influence the study transect and attempt to relate functional soil-borne metabolic activities. community data to specific environmental factors Although some anecdotal evidence exists regard- present in these unique environments. ing C- and N-cycling in Antarctic ecosystems, detailed and systematic data are still mostly lacking. Indeed, the majority of studies addressing polar Materials and methods N- and C-cycles have chiefly involved general process and pool measurements, without regard for Sampling sites the microbial communities responsible for these During the austral summer of 2003–2004, 2 Â 2m processes. Realistic in situ process measurement for plots were established at the following sites (see such remote, low activity, extreme environments are Figure 1 for a map): Falklands Islands (cool especially difficult to obtain. Within the study temperate zone; 511S591W), Signy Island (South presented here, we propose that microarray-based Orkney Islands, maritime Antarctic; 601430S functional gene analysis of the microbial commu- 451380W) and Anchorage Island (near Rothera nities responsible for key nutrient cycle functions research station, Antarctic Peninsula; 671340S might offer an avenue to gain insight into the 681080W).