Quantitative analysis of nifH genes and transcripts from aquatic environments

The availability of fixed inorganic nitrogen often plays a fundamental role in regulating primary production in both aquatic and terrestrial ecosystems. Because biological nitrogen fixation is an important source of nitrogen in marine environments, the study of N2-fixing microorganisms is of fundamental importance to our understanding of global nitrogen and carbon cycles. Quantitative molecular tools have made it possible to examine uncultivated N2-fixing microorganisms directly in the environment. Currently, we are using quantitative polymerase chain reaction (PCR; Q-PCR) and quantitative reverse transcriptase PCR (Q-RT-PCR) to study the ecology and gene expression of N2-fixing bacteria in aquatic environments. Using these methods, we discovered that specific estuarine diazotrophs have distinct nonrandom distributions and that some diazotrophs in the open ocean have different diel patterns of nifH gene expression. This chapter describes briefly our 5' nuclease assay protocols for Q-PCR and Q-RT-PCR of nifH gene fragments in environmental samples and discusses some important methodological considerations for the quantitative molecular examination of microbes in aquatic environments.