Nitrogen fixation: Nitrogenase genes and gene expression

This chapter discusses nitrogenase genes and gene expressions. The primers used for nifH amplification, the methods used to extract genomic DNA and mRNA, the alignment and analysis of nifH sequences, and the RT-PCR protocol are described. Biological nitrogen fixation is the enzymatic reduction of atmospheric dinitrogen to ammonium.

The conventional nitrogenase enzyme is encoded by the nifHDK genes, which are in contiguous arrangement within the genome. Alternative nitrogenases (alternative and second alternative) also contain nifH, but contain a third protein in the counterpart to the Mo protein, which is encoded by nifG (nifDGK).

Nitrogenase genes can be detected and characterized by amplification from environmental samples using the polymerase chain reaction (PCR). Amplification of nitrogenase genes indicates that nitrogen-fixing microorganisms are present, but not whether or not they are actively fixing nitrogen. By coupling, the PCR assay with reverse transcription (RT-PCR) microorganisms that are actively expressing the nitrogenase enzyme can be detected.

Once genes are amplified, the diversity of sequences can be determined by a number of means, including cloning and sequencing of individual amplification products..