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

Abstract

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.

Figure 1

Overview of expression profiling analysis. (A) Venn diagram showing the number of genes differentially regulated under nitrogen fixation (red arrow) and ammonium shock conditions (blue arrow). The bold circle indicates the core subset of nitrogen fixation-inducible genes. The dotted circle indicates the Pseudomonas core genome. The numbers of upregulated and downregulated genes found in the Pseudomonas core genome are shown in parentheses. The number of genes induced specifically under nitrogen fixation conditions is underlined. (B) Functional categories of the core subset of nitrogen fixation, condition-induced genes.

Figure 2

Identification of an expression island in the A1501 genome that supports nitrogen fixation. (A) Schematic representation of the regions in the A1501 chromosome with upregulated and downregulated gene expression. Upregulated (red lines) and downregulated (blue lines) genes under nitrogen fixation and ammonium shock conditions. The 49-kb nif gene expression island (red bar) is located in the chromosome (from PST1302 to PST1359). (B) Predicted operons (black arrows) with conserved RpoN and NifA promoters. The open and filled boxes represent the putative σ⁵⁴-dependent promoter and upstream activator sequences, respectively.

Figure 3

Unrooted neighbor-joining phylogenetic tree of the A1501 PnfA and related proteins from other bacteria. Bootstrap values based on 1,000 replications are listed as percentages at branching points. Bar, 0.1 substitutions per position. The sequence abbreviations are given in parentheses.

Figure 4

Physical organization of the A1501 pnfA cluster and comparisons with equivalent clusters from other diazotrophic strains. The numbers underneath the arrows indicate the percentage of amino acid sequence identity between the A1501 pnfA product and its homologs.

Figure 5

Intergenic region between nifA and pnfA and construction of the pnfA non-polar mutant A1325. The open and filled boxes represent the putative σ⁵⁴-dependent promoter and upstream activator sequences, respectively. The inverted triangle indicates the location of the mutations inserted into pnfA via homologous suicide plasmid integration.

Figure 6

Phenotypic analyses of the mutation inserted into the pnfA gene. (A) Quantitative RT-PCR analysis of pnfA expression in the wild type A1501, nifA mutant A1506, and rpoN mutant A1550 strains under nitrogen fixation conditions. (B) Quantitative RT-PCR analysis of nifH expression in the wild type A1501 and mutant A1325 strains under nitrogen fixation conditions. (C) The effect of different oxygen concentrations on nitrogenase activity in the wild type A1501 and mutant A1325 strains. (D) Nitrogenase activity in the wild type A1501 and mutant A1325 strains grown under anaerobic conditions with or without 1 mM nitrate. * The nitrogenase activity of the mutant strain is expressed as a percentage (%) of the wild type A1501 activity. Results shown are representative of four (C) or three (D) independent experiments.