Abstract
The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.
Highlights
Nitrogen-fixing microorganisms are globally significant in that they provide the only natural biological source of fixed nitrogen in the biosphere
We observe that nucleotide positions near the beginning and end of the nifH gene alignment are underrepresented in sequence databases relative to nucleotide positions in the middle of the gene alignment (Figure 1)
This problem occurs because a majority of nifH sequences have been generated using PCR primers that bind to conserved nucleotide positions found within the nifH gene
Summary
Nitrogen-fixing microorganisms are globally significant in that they provide the only natural biological source of fixed nitrogen in the biosphere. These organisms enzymatically transform dinitrogen gas from the atmosphere into ammonium equivalents needed for biosynthesis of essential cellular macromolecules. Nitrogenfixing bacteria are diverse, and most of the known taxa have not yet been cultivated in the laboratory [1]. Nitrogen fixation is carried out by the nitrogenase enzyme whose multiple subunits are encoded by the genes nifH, nifD, and nifK (as reviewed in [2]). NifH (encoding the nitrogenase reductase subunit) is the most sequenced and has become the marker gene of choice for researchers studying the phylogeny, diversity, and abundance of nitrogen-fixing microorganisms. Many PCR primers have been developed to target the nifH gene with the purpose of amplifying this gene sequence from environmental samples
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