Abstract
BackgroundThe model endophyte Azoarcus sp. strain BH72 is known to contribute fixed nitrogen to its host Kallar grass and also expresses nitrogenase genes endophytically in rice seedlings. Availability of nitrogen is a signal regulating the transcription of nitrogenase genes. Therefore, we analysed global transcription in response to differences in the nitrogen source.Methodology/Principal FindingsA DNA microarray, comprising 70-mer oligonucleotides representing 3989 open reading frames of the genome of strain BH72, was used for transcriptome studies. Transcription profiles of cells grown microaerobically on N2 versus ammonium were compared. Expression of 7.2% of the genes was significantly up-regulated, and 5.8% down-regulated upon N2 fixation, respectively. A parallel genome-wide prediction of σ54-type promoter elements mapped to the upstream region of 38 sequences of which 36 were modulated under the N2 response. In addition to modulation of genes related to N2 fixation, the expressions of gene clusters that might be related to plant-microbe interaction and of several transcription factors were significantly enhanced. While comparing under N2-fixation conditions the transcriptome of wild type with a nifLA− insertion mutant, NifA being the essential transcriptional activator for nif genes, 24.5% of the genome was found to be affected in expression. A genome-wide prediction of 29 NifA binding sequences matched to 25 of the target genes whose expression was differential during microarray analysis, some of which were putatively negatively regulated by NifA. For selected genes, differential expression was corroborated by real time RT-PCR studies.Conclusion/SignificanceOur data suggest that life under conditions of nitrogen fixation is an important part of the lifestyle of strain BH72 in roots, as a wide range of genes far beyond the nif regulon is modulated. Moreover, the NifA regulon in strain BH72 appears to encompass a wider range of cellular functions beyond the regulation of nif genes.
Highlights
Biological nitrogen fixation, involving the enzymatic conversion of atmospheric nitrogen to ammonia by nitrogenase, is an important process to maintain soil fertility and life on earth, counterbalancing loss of nitrogen e.g. by denitrification
The N2 response of Azoarcus sp. strain BH72 resulted in upregulation of 144 genes and down-regulation of 174 genes, independent of the NifA regulon accounting to 61% of the response
Outside the nif related genes there is a significant upregulation of genes for hypothetical proteins and non-nif related gene clusters and down regulation of several general metabolismrelated genes under N2 fixation in strain BH72
Summary
Biological nitrogen fixation, involving the enzymatic conversion of atmospheric nitrogen to ammonia by nitrogenase, is an important process to maintain soil fertility and life on earth, counterbalancing loss of nitrogen e.g. by denitrification. It is catalyzed by the two-component nitrogenase complex restricted to prokaryotes, the reaction being oxygen-sensitive demanding high amounts of energy and reductants. Nitrogen-fixing microorganisms encompass a broad habitat range from free living forms in soils and water to endophytic association with grasses or in rootnodule symbioses with legumes. They have evolved sophisticated regulatory networks that respond to multiple environmental cues [1]. We analysed global transcription in response to differences in the nitrogen source
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