ABSTRACT
The nifH gene is a widely used molecular proxy for studying nitrogen fixation. Phylogenetic classification of nifH gene sequences is an essential step in diazotroph community analysis that requires a fast automated solution due to increasing size of environmental sequence libraries and increasing yield of nifH sequences from high-throughput technologies. A novel approach to rapidly classify nifH amino acid sequences into well-defined phylogenetic clusters that provides a common platform for comparative analysis across studies is presented. Phylogenetic group membership can be accurately predicted with decision tree-type statistical models that identify and utilize signature residues in the amino acid sequences. Our classification models were trained and evaluated with a publicly available and manually curated nifH gene database containing cluster annotations. Model-independent sequence sets from diverse ecosystems were used for further assessment of the models' prediction accuracy. The utility of this novel sequence binning approach was demonstrated in a comparative study where joint treatment of diazotroph assemblages from a wide range of habitats identified habitat-specific and widely-distributed diazotrophs and revealed a marine - terrestrial distinction in community composition. Our rapid and automated phylogenetic cluster assignment circumvents extensive phylogenetic analysis of nifH sequences; hence, it saves substantial time and resources in nitrogen fixation studies.
ABSTRACT
A dataset of 87 020 nifH reads and 16 782 unique nifH protein sequences obtained over 2 years from four locations across a gradient of agricultural soil types in Argentina were analysed to provide a detailed and comprehensive picture of the diversity, abundance and responses of the N2 -fixing community in relation to differences in soil chemistry and agricultural practices. Phylogenetic analysis revealed an expected high proportion of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria, mainly relatives to Bradyrhizobium and Methylosinus/Methylocystis, but a surprising paucity of Gammaproteobacteria. Analysis of variance and stepwise regression modelling suggested location and treatment-specific influences of soil type on diazotrophic community composition and organic carbon concentrations on nifH diversity. nifH gene abundance, determined by quantitative real-time polymerase chain reaction, was higher in agricultural soils than in non-agricultural soils, and was influenced by soil chemistry under intensive crop rotation but not under monoculture. At some locations, sustainable increased crop yields might be possible through the management of soil chemistry to improve the abundance and diversity of N2 -fixing bacteria.
