Qin Li

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH ₄ ⁺ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH ₄ ⁺ ) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH ₄ ⁺ ), nitrogenase activity is reestablished when very high levels of NH ₄ ⁺ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH ₄ ⁺ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH ₄ ⁺ (10 mM). Among the differentially expressed genes, ald1 , encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH ₄ ⁺ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH ₄ ⁺ , inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe–S cluster in diazotrophs. However, nifS and nifU are not found in N₂-fixing Paenibacillus strains, and the mechanisms involved in Fe–S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe–S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe–S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe–S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNA^ser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Application of diazotrophs (N₂-fixing microorganisms) can decrease the overuse of nitrogen (N) fertilizer. Until now, there are few studies on the effects of diazotroph application on microbial communities of major crops. In this study, the diazotrophic and endospore-forming Paenibacillus triticisoli BJ-18 was inoculated into maize soils containing different N levels. The effects of inoculation on the composition and abundance of the bacterial, diazotrophic and fungal communities in the rhizosphere and root/shoot endosphere of maize were evaluated by sequencing the 16S rRNA, nifH gene and ITS (Inter Transcribed Spacer) region. P. triticisoli BJ-18 survived and propagated in all the compartments of the maize rhizosphere, root and shoot. The abundances and diversities of the bacterial and diazotrophic communities in the rhizosphere were significantly higher than in both root and shoot endospheres. Each compartment of the rhizosphere, root and shoot had its specific bacterial and diazotrophic communities. Our results showed that inoculation reshaped the structures of the bacterial, diazotrophic and fungal communities in the maize rhizosphere and endosphere. Inoculation reduced the interactions of the bacteria and diazotrophs in the rhizosphere and endosphere, while it increased the fungal interactions. After inoculation, the abundances of Pseudomonas, Bacillus and Paenibacillus in all three compartments, Klebsiella in the rhizosphere and Paenibacillus in the root and shoot were significantly increased, while the abundances of Fusarium and Giberella were greatly reduced. Paenibacillus was significantly correlated with plant dry weight, nitrogenase, N₂-fixing rate, P solubilization and other properties of the soil and plant.

Exopolysaccharides (EPS) are of high significance in bacterial biofilm formation. However, the effects of EPS cluster(s) on biofilm formation in Paenibacillus species are little known. In this study, we have shown that Paenibacillus polymyxa WLY78, a N₂-fixing bacterium, can form biofilm. EPS is the major component of the extracellular matrix. The genome of P. polymyxa WLY78 contains two putative gene clusters (designated pep-1 cluster and pep-2 cluster). The pep-1 cluster is composed of 12 putative genes (pepO-lytR) co-located in a 13 kb region. The pep-2 cluster contains 17 putative genes (pepA-pepN) organized as an operon in a 20 kb region. Mutation analysis reveals that the pep-2 cluster is involved in EPS biosynthesis and biofilm formation. Disruption of the pep-2 cluster also leads to the enhancement of motility and change of the colony morphology. In contrast, disruption of the pep-1 cluster does not affect EPS synthesis or biofilm formation. More importantly, the biofilm allowed P. polymyxa WLY78 to fix nitrogen in aerobic conditions, suggesting that biofilm may provide a microaerobic environment for nitrogenase synthesis and activity.

BackgroundBiological nitrogen fixation catalyzed by nitrogenase is a high energy-intensive process, and thus nitrogenase synthesis and activity are inhibited by ammonium (NH₄⁺). Microorganism fix nitrogen at high ammonium (30-300 mM) concentration has not been reported before.ResultsPaenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph (N₂-fixing microorganism, showed nitrogenase activities not only in low (0-4 mM) concentration of NH₄⁺, but also in high (30-300 mM) concentration of NH₄⁺, no matter whether the cells of this bacterium were grown in flask or in fermentor on scale cultivation. qRT-PCR and western blotting analysis supported that Fe protein and MoFe protein were synthesized under both low (0-4 mM) and high (30-300 mM) concentration of NH₄⁺. Liquid chromatography-mass spectrometry（LC-MS）analysis revealed that MoFe protein purified form cultures grown in nitrogen-limited condition or nitrogen-excess condition was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and -excess conditions were active.ConclusionsOur results indicate that N₂ fixation occurs in presence of high (30-300 mM) concentration of NH₄⁺ in P. sabinae T27. Nitrogen fixation under both low and high concentration of NH₄⁺ was catalyzed by the same nitrogenases and the Fe protein was encoded by both nifH and nifH2. Our study will provide a clue for studying the mechanisms on nitrogen fixation in presence of the high concentration of NH₄⁺.

Nitrogen is one of the most important nutrients for plant growth. To enhance crop productivity, chemical nitrogen fertilizer is commonly applied in agriculture. Biological nitrogen fixation, the conversion of atmospheric N₂ to NH₃, is an important source of nitrogen input in agriculture and represents a promising substitute for chemical nitrogen fertilizers. However, nitrogen fixation is only sporadically distributed within bacteria and archaea (diazotrophs). Thus, many biologists hope to reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host, with the final aim of developing N₂‐fixing cereal crops. With the advent of synthetic biology and a deep understanding of the fundamental genetic determinants necessary to sustain nitrogen fixation in bacteria, much progress has been made toward this goal. Transfer of native and refactored nif (nitrogen fixation) genes to non‐diazotrophs has been attempted in model bacteria, yeast, and plants. Specifically, nif genes from Klebsiella oxytoca, Azotobacter vinelandii, and Paenibacillus polymyxa have been successfully transferred and expressed in Escherichia coli, Saccharomyces cerevisiae, and even in the tobacco plant. These advances have laid the groundwork to enable cereal crops to “fix” nitrogen themselves to sustain their growth and yield.

Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. Genomic sequence analysis has revealed that a compact nif (nitrogen fixation) gene cluster comprising 9–10 genes nifBHDKENX(orf1)hesAnifV is conserved in diazotrophic Paenibacillus species. The evolution and function of the orf1 gene within the nif gene cluster of Paenibacillus species is unknown. In this study, a careful comparison analysis of the compositions of the nif gene clusters from various diazotrophs revealed that orf1 located downstream of nifENX was identified in anaerobic Clostridium ultunense, the facultative anaerobic Paenibacillus species and aerobic diazotrophs (e.g., Azotobacter vinelandii and Azospirillum brasilense). The predicted amino acid sequences encoded by the orf1 gene, part of the nif gene cluster nifBHDKENXorf1hesAnifV in Paenibacillus graminis RSA19, showed 60–90% identity with those of the orf1 genes located downstream of nifENX from different diazotrophic Paenibacillus species, but shared no significant identity with those of the orf1 genes from different taxa of diazotrophic organisms. Transcriptional analysis showed that the orf1 gene was expressed under nitrogen fixation conditions from the promoter located upstream from nifB. Mutational analysis suggested that the orf1 gene functions in nitrogen fixation in the presence of a high concentration of O₂.