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

Biological nitrogen fixation, a process utilized only by certain prokaryotes, is catalyzed by a two-component nitrogenase complex [1]. Nitrogen-fixing microorganisms colonize a wide variety of habitats and can be found free-living in soils and water, in association with grasses, or in root-nodule symbioses with legumes. Consequently, they have evolved sophisticated regulatory networks that respond to multiple environmental cues [2]. Fixed nitrogen, such as ammonium, is one of the most important environmental signals that regulate nitrogen fixation. Regulation of nif gene expression has been most extensively studied in diazotrophic proteobacteria [3]. It is becoming increasingly apparent that the genes necessary for nitrogen fixation in many diazotrophs have common structures and functions. However, the mechanisms by which cellular nitrogen levels are sensed and nitrogen signals are transmitted can vary considerably among different nitrogen-fixing bacteria [4].

The availability of diazotrophic genome data can provide insights into the gene regulatory network of nitrogen-fixing bacteria. Whole-genome microarray platforms are appropriate for the study on the environmental responses and the global regulation of diazotrophs. For example, this approach was used to analyze the genes that regulate nitrogen fixation and Fe (III) reduction in Geobacter sulfurreducens [5]; analyze the general stress responses of A. vinelandii during the carbon and nitrogen diauxic shift [6]; perform transcriptome analysis of Sinorhizobium meliloti during symbiosis [7]; identify expression islands clustered on the symbiosis island of the Mesorhizobium loti genome [8]; and perform transcriptomic studies of Bradyrhizobium japonicum in the bacteroid state [9] and during chemoautotrophic growth [10]. In addition, Rediers et al. [11] demonstrated the successful application of a dap-based in vivo expression technology selection strategy to identify P. stutzeri A1501 genes that are switched on during rice root colonization and off during free-living growth. 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]. Collectively, 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.

A1501 is a versatile soil bacterium that exhibits the unusual feature, for a Pseudomonas, of being capable of nitrogen fixation, and it is capable of endophytic association with rice plants [11, 13–19]. This strain has received particular attention because of its specific metabolic properties, which include denitrification under anaerobic conditions, nitrification under aerobic conditions, and nitrogen fixation under microaerophilic conditions [14]. The association of A1501 with crop plants is a promising model system for the study of plant-microbe-soil interactions in the rhizosphere. The response of bacteria to fixed nitrogen has often been studied after short-term exposure to excess ammonium concentrations, which we refer to as ammonium shock. Although the genetic and physiological responses to fixed nitrogen concentrations have been well studied in A1501 and other diazotrophic strains, the response of nitrogen-fixing bacteria to ammonium shock has not yet been examined using DNA microarray analysis. In an effort to better understand the transcriptional regulation of nif gene expression, we took advantage of the availability of the complete genome sequence of A1501 to construct a genomic array system that we used to profile the global transcription of A1501 under nitrogen fixation and ammonium shock conditions. We identified 255 upregulated genes under nitrogen fixation conditions, 166 of which were found to be collectively downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in Azoarcus sp. BH72 and A. vinelandii AvoP. Finally, we identified a new gene, termed pnfA, which might play a role in the control of nitrogenase activity.

Nitrogen fixation is tightly regulated at the transcriptional and post-translational levels [2]. The first checkpoint occurs at the level of the nif-specific NifA-NifL interaction, which is controlled by the Ntr systems [28]. The second checkpoint occurs upon reversible inactivation of nitrogenase by dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG) [29]. In many diazotrophic γ-Proteobacteria, such as in K. pneumoniae, A. vinelandii, and P. stutzeri, the σ⁵⁴-dependent activator NifA activates the expression of all the nif genes, and the anti-activator NifL controls NifA activity via protein-protein interactions in response to fixed nitrogen status [18, 30, 31]. Different regulatory mechanisms at the transcriptional level have been documented in several diazotrophs, such as in the α-Proteobacteria A. brasilense, which lacks nifL [32]. PII proteins are key signal transduction proteins involved in the general nitrogen regulation system and are found in diazotrophic Proteobacteria as two paralogous gene copies, glnB and glnK [19, 33–35]. In K. pneumoniae, the GlnB protein controls the activity of the NtrB-NtrC regulatory system; in turn, phosphorylation of NtrC controls the expression of the alternative PII protein GlnK, as well as of the NifL and NifA proteins [28]. A1501 carries a single copy of the glnK gene, similar to A. vinelandii, but lacks glnB, which is present in K. pneumoniae and A. brasilense [28, 32]. GlnK can interact with NifL to promote the formation of a GlnK-NifL-NifA ternary complex in A. vinelandii and P. stutzeri [19, 36]. Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in A1501 using a whole-genome DNA microarray. The results show that among the nitrogen fixation-inducible genes in A1501, including those involved in the Ntr and nif-specific regulatory systems, 95 have orthologs in Azoarcus sp. BH72 and A. vinelandii AvoP. This type of detailed global analysis had not been done before. Our results suggest the existence of similar regulatory mechanisms that control expression of nitrogen fixation genes in the three most closely related diazotrophic strains. Moreover, although nitrogenase on-off switching was previously reported [13], DraT and DraG (known to control nitrogenase ADP ribosylation) have not been found in A1501, suggesting that it possesses an inactivation mechanism different from that reported in Rhodospirillum rubrum [37], Azoarcus [38], and A. brasilense [39].

Horizontal gene transfer plays an important role in the evolution of the nitrogen fixation system [40–43]. Baar et al. [44] observed that a gene cluster containing the nif genes within the genome of Wolinella succinogenes exhibits a high degree of synteny to the cyanobacterial nif gene cluster, implying that the nif genes may have been acquired by gene transfer. A so-called "symbiosis island" was initially identified in M. loti ICMP3153 as a transmissible 500-kb DNA element, which contains all the genes that are likely to be required for Nod factor, nitrogen fixation, and island transfer [45]. After years of controversy, it is now established that several strains of Pseudomonas species can fix nitrogen [14]. However, the sporadic occurrence of functional nif genes in Pseudomonas spp. raises the question of their origin [16]. Based on genomic comparisons, the number of genes previously recognized as being part of the Pseudomonas core genome may be limited to 1,997 [20]. In the present study, we found that more than 70% of nitrogen fixation-inducible genes, including those within the nitrogen fixation island, are absent from the Pseudomonas core genome. Only 24 nitrogen fixation-inducible genes, such as the ntr genes, belong to the Pseudomonas core genome (Table 3). Our data on global transcriptional profiling strongly suggest that the A1501 nif region is not only a genomic island but also an expression island. The ntr system exists in almost all organisms, whereas the nifLA regulatory system is highly specific for nitrogen-fixing bacteria [33, 34]. Apparently, A1501 has its own ntr systems and acquired a nif-specific regulatory system from a diazotrophic ancestor during its evolution. Consequently, the acquisition of nif genes and cooperative control of two regulatory systems of different evolutionary origins confer A1501 with the unusual capability to fix nitrogen. However, the consequences of nif island acquisition for the host and for island function upon entrance into a new global regulatory network have not been directly studied.

We further compared the organization of the A1501 nif cluster with that of K. pneumoniae, which exhibits the highest organization of nif genes identified to date. The high conservation of the core nif genes between the two diazotrophic strains suggests that they were acquired from a common ancestor. Interestingly, we found that many additional genes, such as pnfA, which is associated with the core nif genes, are found in A1501 but not exist in K. pneumoniae. From these facts, we suppose that lateral transfer of the nif genes from a common ancestor and subsequent acquisition of additional genes occurred to for bacterial adaptation to the physiology of their respective hosts. In nitrogen-free semisolid medium, A1501 formed a pellicle under the surface and exhibited a high level of nitrogenase activity, suggesting that nitrogen fixation occurs at low oxygen tension [13]. Under anaerobic conditions, nitrate can accept the terminal electron transferred from the respiratory chain, and the energy generated during this process supports nitrogen fixation in A1501 [46]. However, the genes responsible for the transport of electrons to nitrogenase and for the oxygen protection mechanisms in A1501 have yet to be determined.

The objective of this study was to explore the utility of transcriptional profiling as a screening method for identifying previously uncharacterized genes relevant for the nitrogen fixation processes. In addition, we focused on elucidating the roles of newly characterized genes in controlling the expression or activity of nitrogenase. We first identified a set of hypothetical genes that are induced under nitrogen fixation conditions and subsequently carried out a functional analysis of these genes. Consequently, we identified a new gene, referred to as pnfA, which is chromosomally linked to nifHDK. This new gene has the following characteristics: (i) like all known nif genes, it is upregulated exclusively under nitrogen fixation conditions in a NifA-σ⁵⁴-dependent manner; (ii) it encodes a 31.7-kDa protein with high similarity to hypothetical proteins present only in nitrogen-fixing bacteria, suggesting that it is a diazotroph-specific protein; (iii) its inactivation results in the inability of bacteria to use nitrate as a terminal electron acceptor for supporting nitrogen fixation under anaerobic conditions. However, the pnfA mutant can grow on nitrogen-free semisolid medium, indicating that it has a Nif⁺ phenotype (data not shown). Furthermore, a similarity search revealed that a portion of the amino acid sequence of the PnfA protein (residues 176 to 240) has approximately 31% identity to equivalent regions from the UspA domain-containing protein of Mycobacterium sp. JLS and the electron transfer flavoprotein alpha subunit-like protein of Methylobacterium nodulans ORS 2060. This result suggests that PnfA may play a role in the transport of electrons to nitrogenase or in the regulation of oxygen protection mechanisms. This finding is also supported by the observation that homologues of PnfA are present in many associative or endophytic nitrogen-fixing bacteria, such as A1501, Azoarcus sp. BH72, and A. vinelandii AvoP, which usually utilize different strategies for meeting the energy demand of nitrogen fixation and protecting nitrogenase from oxygen. Further studies on the expression and regulation of nitrogen fixation-inducible genes in A1501 will provide useful insights into how the bacterium responds to environmental cues and how it adapts to rhizosphere environments.

In the present study, we performed a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in P. stutzeri A1501 using a whole-genome DNA microarray. In total, 166 genes were upregulated under nitrogen fixation conditions and rapidly downregulated as early as 10 min after ammonium shock, suggesting the existence of a regulon of genes that are induced specifically under nitrogen fixation conditions. Strikingly, we found that a 49-kb cluster of all nif and associated genes was markedly upregulated as an expression island, whereas genes outside the island were downregulated under nitrogen fixation conditions. Furthermore, we identified a new NifA-σ⁵⁴-dependent gene, termed pnfA, which is located within the expression island and may be involved in the nitrogen fixation processes. The implications of our functional and evolutionary analyses of the nitrogen fixation island and the pnfA gene in root-associated and nitrogen-fixing bacteria are discussed.