Abstract
BackgroundBiological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation.ResultsHere we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-σ54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions.ConclusionsBased on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif-specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.
Highlights
Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen
Short exposure of the bacteria to excess ammonia induced the expression of various genes for translation apparatus, including forty-four genes encoding many ribosomal proteins, three genes encoding the DNA-directed RNA polymerase, and five genes encoding protein translation factors and the ribosome-binding factor
We found that 166 genes were upregulated under nitrogen fixation conditions but were rapidly downregulated as early as 10 min after ammonium shock, indicating a regulon of nitrogen fixation-inducible genes in A1501
Summary
Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. The availability of diazotrophic genome data can provide insights into the gene regulatory network of nitrogen-fixing bacteria. An analysis of NifA-dependent transcription patterns using a competitive hybridization method led to the identification of three novel NifA-regulated genes (nrgA, nrgB, and nrgC) in the symbiotic region of B. japonicum [12]. These data suggest that a large fraction of the proteins potentially involved in the control of nitrogen fixation remain uncharacterized. We believe that a more complete characterization of these genes will shed light on the underlying molecular mechanisms of nitrogen fixation and contribute to our understanding of the evolution of nitrogen-fixing bacteria
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