Functional marker genes for identification of sulfate-reducing prokaryotes

Sulfate-reducing prokaryotes (SRPs) exploit sulfate as an electron acceptor for anaerobic respiration and exclusively catalyze this essential step of the world's sulfur cycle. Because SRPs are found in many prokaryotic phyla and are often closely related to non-SRPs, 16S rRNA gene-based analyses are inadequate to identify novel lineages of this guild in a cultivation-independent manner. This problem can be solved by comparative sequence analysis of environmentally retrieved gene fragments of the dissimilatory (bi)sulfite (dsrAB) and adenosine-5'-phosphosulfate reductases (apsA), which encode key enzymes of the SRP energy metabolism. This chapter provides detailed protocols for the application of these functional marker molecules for SRP diversity surveys in the environment. Data from the analysis of dsrAB sequence diversity in water samples from the Mariager Fjord in northeast Denmark are presented to illustrate the different steps of the protocols. Furthermore, this chapter describes a novel gel retardation-based technique, suitable for fingerprinting of the approximately 1.9-kb-large dsrAB polymerase chain reaction amplification products, which efficiently increases the chance of retrieving rare and novel dsrAB sequence types from environmental samples.