A nifH-based Oligonucleotide Microarray for Functional Diagnostics of Nitrogen-fixing Microorganisms
- 264 Downloads
Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.
KeywordsClone Library Wild Rice Terminal Restriction Fragment Length Polymorphism Microarray Hybridization nifH Gene
This work was linked to the CAG supported by the Land Bremen and the German Federal Ministry of Education and Research (BMBF) (grant 0311833 A, TP 14 to B. R.-H.), and was also supported by a grant of the BMBF under the BIOLOG framework to B. R.-H. and T. H. (grant no. 01LC0021). For the sequences of the probe set of the nifH microarray, an application for a patent has been filed by the University of Bremen (PCT/EP2005/008150 and is available at EBI W02006013052).
- 19.Nicholas, KB, Nicholas, HBJ (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. http://www.cris.com/~ketchup/genedoc.shtml
- 25.Reinhold-Hurek, B, Hurek, T (2000) Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and species, Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov, and Azonexus funguphilus gen. nov., sp. nov. Int J Syst Evol Microbiol 50: 649–659PubMedGoogle Scholar
- 26.Reinhold-Hurek, B, Hurek, T, Gillis, M, Hoste, B, Vancanneyt, M, Kersters, K, De Ley, J (1993) Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth) and description of two species Azoarcus indigens sp. nov. and Azoarcus communis sp. nov. Int J Syst Bacteriol 43: 574–584CrossRefGoogle Scholar