Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501.
BACKGROUND
Biological 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.


RESULTS
Here 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-sigma54-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.


CONCLUSIONS
Based 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.
Yan
Yongliang
Y
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Crop Biotechnology, Ministry of Agriculture, Beijing 100081, China.
Ping
Shuzhen
S
Peng
Junping
J
Han
Yunlei
Y
Li
Liang
L
Yang
Jian
J
Dou
Yuetan
Y
Li
Yan
Y
Fan
Huili
H
Fan
Ying
Y
Li
Danhua
D
Zhan
Yuhua
Y
Chen
Ming
M
Lu
Wei
W
Zhang
Wei
W
Cheng
Qi
Q
Jin
Qi
Q
Lin
Min
M
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D005810:Multigene Family; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000378:metabolism; D010802:Phylogeny; D018517:Plant Roots / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044223:Pseudomonas stutzeri / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics
D001426:Bacterial Proteins; D000644:Quaternary Ammonium Compounds; D014157:Transcription Factors; D009591:Nitrogenase