Development of PCR primers for community studies of bacterial hydrogenases in environmental samples

Abstract: Soil microorganisms interact with plants in ways that affect nutrient availability, hormonal regulation and often lead to improved plant growth. In the N2-fixing root nodule symbiosis between legume plants and bacteria generally called rhizobia the plant gains access to atmospheric nitrogen. In that process H2 is released from the N2-fixing rhizobium to the surrounding plant and soil and is used by the microbial community as energy source. The goal of this master’s thesis was to design PCR primers targeting the gene for the large subunit of the uptake hydrogenase (hupL) that could be used to identify and quantify the part of that community that is able to use H2. Protein sequences of uptake hydrogenases from three phyla (Proteobacteria, Firmicutes and Actinobacteria) known to interact with plant roots were used for phylogenetic analysis and for designing degenerate primers. Sets of primers for obtaining short and long PCR products were designed for each of the three phyla using the software iCODEHOP. The designed primers were verified against their gene sequences, manually edited and controlled for their quality. Using PCR analysis it was possible to generate PCR products of the expected size of 310 to 330 bp using the primers specific for Proteobacteria and Actinobacteria and an environmental DNA sample as the template. Further studies will show identities and diversity of the hupL sequences and the wider usefulness of the designed primers.