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A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms.

Lei Zhang¹, Thomas Hurek, Barbara Reinhold-Hurek

¹Laboratory of General Microbiology, Center of Applied Gene Sensoric, University of Bremen, P.O. Box 33 04 40, D-28334, Bremen, Germany.

Microbial Ecology

|December 23, 2006

Summary

A new microarray tool rapidly monitors nitrogen-fixing bacteria by targeting the nifH gene. This method reveals that only a small fraction of detected diazotrophs are actively fixing nitrogen in wild rice roots.

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Area of Science:

Microbiology
Molecular Ecology
Biogeochemistry

Background:

Nitrogen fixation is a crucial biogeochemical process performed exclusively by prokaryotes.
The nitrogenase gene (nifH) is vital for phylogenetic and diversity studies of nitrogen-fixing organisms.
Environmental samples often contain nifH fragments from uncultivated microorganisms.

Purpose of the Study:

To develop a rapid, oligonucleotide microarray tool for monitoring nitrogen-fixing diazotrophic populations.
To base the microarray design on nifH sequences from uncultivated prokaryotes.
To analyze diazotroph diversity and activity in wild rice roots.

Main Methods:

Development of a nifH-based diagnostic microarray using specific oligonucleotides.
Validation of the microarray with reference strains, environmental clones, and artificial mixtures.
Application of the microarray to analyze DNA (diversity) and mRNA (activity) in wild rice root samples.

Main Results:

The nifH microarray effectively monitors diazotrophic populations across environments.
Analysis of wild rice roots showed that only a subset of present diazotrophs were actively fixing nitrogen.
The microarray is reproducible and semiquantitative for assessing diazotroph abundance and activity.

Conclusions:

The developed nifH microarray is a valuable tool for rapid environmental monitoring of diazotrophs.
It allows for comparative analysis of diazotrophic prokaryote diversity and activity.
A refined microarray with 194 probes covers over 90% of database nifH sequences.