Paenibacillus polymyxa has widely been studied as a model of plant-growth promoting rhizobacteria (PGPR). Here, the genome sequences of 9 P. polymyxa strains, together with 26 other sequenced Paenibacillus spp., were comparatively studied. Phylogenetic analysis of the concatenated 244 single-copy core genes suggests that the 9 P. polymyxa strains and 5 other Paenibacillus spp., isolated from diverse geographic regions and ecological niches, formed a closely related clade (here it is called Poly-clade). Analysis of single nucleotide polymorphisms (SNPs) reveals local diversification of the 14 Poly-clade genomes. SNPs were not evenly distributed throughout the 14 genomes and the regions with high SNP density contain the genes related to secondary metabolism, including genes coding for polyketide. Recombination played an important role in the genetic diversity of this clade, although the rate of recombination was clearly lower than mutation. Some genes relevant to plant-growth promoting traits, i.e. phosphate solubilization and IAA production, are well conserved, while some genes relevant to nitrogen fixation and antibiotics synthesis are evolved with diversity in this Poly-clade. This study reveals that both P. polymyxa and its closely related species have plant growth promoting traits and they have great potential uses in agriculture and horticulture as PGPR.

Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.

Beneficial rhizobacteria promote plant growth and protect plants against phytopathogens. Effective colonization on plant roots is critical for the rhizobacteria to exert beneficial activities. How bacteria migrate swiftly in the soil of semisolid or solid nature remains unclear. Here we report that sucrose, a disaccharide ubiquitously deployed by photosynthetic plants for fixed carbon transport and storage, and abundantly secreted from plant roots, promotes solid surface motility (SSM) and root colonization by Bacillus subtilis through a previously uncharacterized mechanism. Sucrose induces robust SSM by triggering a signaling cascade, first through extracellular synthesis of polymeric levan, which in turn stimulates strong production of surfactin and hyper-flagellation of the cells. B. subtilis poorly colonizes the roots of Arabidopsis thaliana mutants deficient in root-exudation of sucrose, while exogenously added sucrose selectively shapes the rhizomicrobiome associated with the tomato plant roots, promoting specifically bacilli and pseudomonad. We propose that sucrose activates a signaling cascade to trigger SSM and promote rhizosphere colonization by B. subtilis. Our findings also suggest a practicable approach to boost prevalence of beneficial Bacillus species in plant protection.

Background Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. ResultsIn order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe–S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytocanifSU (Fe–S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytocanifWZM and nifQ can not increase activity.ConclusionThe combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0442-6) contains supplementary material, which is available to authorized users.

BackgroundDiazotrophic (nitrogen-fixing) Gram-positive and endospore-formed Paenibacillus spp. have potential uses as a bacterial fertilizer in agriculture. The transcriptional analysis of nitrogen fixation in Paenibacillus is lacking, although regulation mechanisms of nitrogen fixation have been well studied in Gram-negative diazotrophs.ResultsHere we report a global transcriptional profiling analysis of nitrogen fixation in Paenibacillus sp. WLY78 cultured under N2-fixing condition (without O2 and NH4+) and non-N2-fixing condition (air and 100 mM NH4+). The nif (nitrogen fixation) gene operon composed of 9 genes (nifBHDKENXhesAnifV) in this bacterium was significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition, indicating that nif gene transcription is strictly controlled by NH4+ and O2. qRT-PCR confirmed that these nif genes were differently expressed. Non-nif genes specifically required in nitrogen fixation, such as mod, feoAB and cys encoding transporters of Mo, Fe and S atoms, were coordinately transcribed with nif genes in N2-fixing condition. The transcript abundance of suf operon specific for synthesis of Fe-S cluster was up-regulated in N2-fixing condition, suggesting that Sul system, which takes place of nifS and nifU, plays important role in the synthesis of nitrogenase. We discover potential specific electron transporters which might provide electron from Fe protein to MoFe protein of nitrogenase. The glnR whose predicted protein might mediate nif transcription regulation by NH4+ is significantly up-regulated in N2-fixing condition. The transcription levels of nitrogen metabolism and anaerobic respiration were also analyzed.ConclusionsThe nif gene operon (nifBHDKENXhesAnifV) in Paenibacillus sp. WLY78 is significantly up-regulated in N2-fixing condition compared to non-N2-fixing condition. Non-nif genes specifically required in nitrogen fixation were also significantly up-regulated in N2-fixing condition. Fur and Fnr which are involved in anaerobic regulation and GlnR which might mediate nif gene transcription regulation by NH4+ were significantly up-regulated in N2-fixing condition. This study provides valuable insights into nitrogen fixation process and regulation in Gram-positive firmicutes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0642-6) contains supplementary material, which is available to authorized users.

Biofertilizer is a good substitute for chemical fertilizer in sustainable agriculture, but its effects are often hindered by drought stress. Super absorbent polymer (SAP), showing good capacity of water absorption and retention, can increase soil moisture. However, limited information is available about the efficiency of biofertilizer amended with SAP. This study was conducted to investigate the effects of synergistic application of SAP and biofertilizers (Paenibacillus beijingensis BJ-18 and Bacillus sp. L-56) on plant growth, including wheat and cucumber. Potted soil was treated with different fertilizer combinations (SAP, BJ-18 biofertilizer, L-56 biofertilizer, BJ-18 + SAP, L-56 + SAP), and pot experiment was carried out to explore its effects on viability of inoculants, seed germination rate, plant physiological and biochemical parameters, and expression pattern of stress-related genes under drought condition. At day 29 after sowing, the highest viability of strain P. beijingensis BJ-18 (264 copies ng−1 gDNA) was observed in BJ-18 + SAP treatment group of wheat rhizosphere soil, while that of strain Bacillus sp. L-56 (331 copies ng−1 gDNA) was observed in the L-56 + SAP treatment group of cucumber rhizosphere soil. In addition, both biofertilizers amended with SAP could promote germination rate of seeds (wheat and cucumber), plant growth, soil fertility (urease, sucrose, and dehydrogenase activities). Quantitative real-time PCR analysis showed that biofertilizer + SAP significantly down-regulated the expression levels of genes involved in ROS scavenging (TaCAT, CsCAT, TaAPX, and CsAPX2), ethylene biosynthesis (TaACO2, CsACO1, and CsACS1), stress response (TaDHN3, TaLEA, and CsLEA11), salicylic acid (TaPR1-1a and CsPR1-1a), and transcription activation (TaNAC2D and CsNAC35) in plants under drought stress. These results suggest that SAP addition in biofertilizer is a good tactic for enhancing the efficiency of biofertilizer, which is beneficial for plants in response to drought stress. To the best of our knowledge, this is the first report about the effect of synergistic use of biofertilizer and SAP on plant growth under drought stress.

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N2-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.

An efficient synthesis of biologically important quinazolinones via using dimethyl sulfoxide as a synthon has been developed. This reaction proceeds smoothly to obtain the corresponding quinazolinones products in good yield under metal free conditions and shows an excellent functional group tolerance. A preliminary mechanistic study indicates that the C2 hydrogen of the synthesized heterocycles come from the dimethyl sulfoxide.