Search Results (37)

Microbially mediated biological nitrogen fixation is pivotal to sustainable agricultural development. However, optimizing nitrogenase activity in native biological nitrogen-fixing bacteria has been hindered by the complexities of genetic manipulation. Heterologous expression has served as a foundational strategy for engineering next-generation nitrogen-fixing microbial agents. In this study, genomic analysis of Paenibacillus polymyxa CR1 revealed an 11 kb nitrogen-fixing (nif) gene cluster. The nif cluster was first synthesized and then assembled using ExoCET technology and finally integrated into the genome of Bacillus subtilis 168 via double-exchange recombination. RT-PCR confirmed the transcription of the nif cluster; however, no nitrogenase activity was detected in the acetylene reduction assay. A promoter replacement strategy (replacing the native promoter with P_veg) enabled B. subtilis to produce active nitrogenase. However, stronger promoters—namely, P₄₃ and P_tp2—did not further enhance nitrogenase activity. This demonstrates that promoter selection requires balancing transcriptional strength with systemic compatibility, particularly for metalloenzymes demanding precise cofactor assembly. This is the first report describing the heterologous expression of the nif gene cluster in B. subtilis, establishing a foundation for engineering high-efficiency nitrogen-fixing biofertilizers. Full article

Rice is a major crop for half of the world’s population, and nitrogen (N) fertilizers play a crucial role in its production. However, imbalanced N fertilizer uses and traditional fertilization practices have led to low nitrogen use efficiency (NUE), increased N footprints, and reduced rice yields and farmers’ income. There are limited studies where the integration of both agronomic and molecular advancements to enhance NUE is discussed, particularly in developing countries. This review highlights novel agronomic and molecular strategies to enhance NUE, rice yields, and profitability, while minimizing environmental impact. The agronomic strategies include the 4R Nutrient Stewardship framework, enhanced efficiency nitrogen fertilizers (EENFs), nano-fertilizers, biochar-based fertilizers, biological N fixation, and sensor-based fertilizer management in major rice-growing countries. The molecular mechanisms focus on N uptake, assimilation, and utilization, highlighting the role of hormones, key genes, transcription factors (TFs), and regulatory pathways. Moreover, we examine promising rice genotypes and cultivars with improved NUE and grain yield. Additionally, this paper offers deep insights into recent advancements in molecular genetics, such as multi-omics approaches (transcriptomics, metabolomics, and metagenomics), the Genome-Wide Association Study (GWAS), Quantitative Traits Loci mapping (QTLs), Single Nucleotide Polymorphisms (SNPs) analysis, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9)-mediated genome editing, which serve as valuable tools for developing rice cultivars with enhanced NUE and grain yield. Full article

Phytobacter palmae WL65, isolated from the rice rhizosphere, was confirmed as P. palmae through whole-genome analysis. WL65 exhibited key plant growth-promoting (PGP) characteristics, including nitrogen fixation (nifA, nifB, nifD, nifE, nifF, nifH, nifJ, nifK, nifL, nifS, nifU, nifW, and nifX), phosphate solubilization (pstA, pstB, pstC, pstS, phnC, phnD, phnE, and phnV), siderophore production (fhuA, fhuB, fhuC, fhuD, fhuF, feoA, feoB, feoC, acrA, acrB, acrE, acrR, and acrZ), and phytohormone biosynthesis (trpA, trpB, trpC, trpE, trpGD, trpR, and trpS). WL65 also contains an enterobactin biosynthetic gene cluster, essential for iron acquisition and enhancing both bacterial survival and plant growth. This study provides the first genomic insights into the PGP characteristics of P. palmae. The application of WL65 in rice cultivation as a biostimulant resulted in effective root colonization, supported by biofilm formation genes (pgaA, pgaB, pgaC), which enhance bacterial adhesion. The treatment significantly improved rice growth, increasing plant height (5.8%), panicle length (10.2%), and seed yield (34.5%). Soil analysis revealed improved nutrient availability, including increased organic matter (21%), phosphorus (38.4%), potassium (29.8%), and calcium (27%) levels. These findings suggest that WL65 is a promising biofertilizer candidate for improving soil fertility and nutrient uptake in sustainable agriculture. Full article

Plants inhabiting environments with suboptimal growth conditions often have a more pronounced capacity to attract and sustain microbial communities that improve nutrient absorption and expand abiotic stress tolerance. Rhodiola rosea L. is a succulent plant of the Crassulaceae family adapted to survive in sandy or rocky soils or dry tundra. The aim of the present study was to investigate the diversity and plant growth-stimulating potential of R. rosea endophytic microbiota. Metataxonomic analysis of the bacterial diversity in the rhizome of R. rosea revealed 108 families. Among these, three families were found exclusively in the core microbiome of 1-year-old plants, while nine families were unique to the core microbiome of mature plants grown in the field for more than 4 years. Seventy-three endophytic bacteria isolates were obtained from the rhizome of R. rosea plants and were assigned into 14 distinct bacterial genera of Firmicutes (26%) or Proteobacteria (74%) phyla. Screening for functional genes related to the nitrogen cycle, phosphorus mineralisation or dissolution, and traits associated with nitrogen fixation (56% of isolates), siderophore production (40%), inorganic phosphorus solubilisation (30%), and production of indole-related compounds (51%) led to the classification of the isolates into 16 distinct clusters. Co-cultivation of 45 selected isolates with germinating Arabidopsis seedlings revealed 18 and 5 isolates that resulted in more than a 20% increase in root or shoot growth, respectively. The study results established the complexity of the succulent R. rosea endophytic microbiome and identified isolates for potential plant growth-stimulating applications. Full article

Two strains, M1 and H32 with nitrogen-fixing ability, were isolated from the rhizospheres of different plants. Genome sequence analysis showed that a nif (nitrogen fixation) gene cluster composed of nine genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) was conserved in the two strains. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains M1 and H32 are members of the genus Paenibacillus. Strains M1 and H32 had 97% similarity in the 16S rRNA gene sequences. Strain M1 had the highest similarity (97.25%) with Paenibacillus vini LAM 0504T in the 16S rRNA gene sequences. Strain H32 had the highest similarity (97.48%) with Paenibacillus faecis TCIP 101062T in the 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain M1 and its closest member P. vini were 78.17% and 22.3%, respectively. ANI and dDDH values between strain H32 and its closest member P. faecis were 88.94% and 66.02%, respectively. The predominant fatty acid of both strains is anteiso-C15:0. The major polar lipids of both strains are DPG (diphosphatidylglycerol) and PG (phosphatidylglycerol). The predominant isoprenoid quinone of both strains is MK-7. With all the phylogenetic and phenotypic divergency, two novel species Paenibacillus haidiansis sp. nov and Paenibacillus sanfengchensis sp. nov are proposed with the type strain M1^T [=GDMCC (Guangdong Culture Collection Centre of Microbiology) 1.4871 = JCM (Japan Collection of Microorganisms) 37487] and with type strain H32^T (=GDMCC 1.4872 = JCM37488). Full article

Biological nitrogen fixation by rhizobia-nodulated legumes reduces the dependence on synthetic nitrogen fertilizers. Identification of locally adapted rhizobia may uncover economically valuable strains for sustainable agriculture. This study investigated the diversity and symbiotic potential of rhizobia associated with three Medicago species from Eastern Romania’s ecosystems. Phenotypic screening ensured that only rhizobial species were retained for molecular characterization. 16S rDNA sequencing clustered the isolates into four distinct groups: Sinorhizobium meliloti, Sinorhizobium medicae, Rhizobium leguminosarum, and Mesorhizobium spp. The chromosomal genes (atpD, glnII, recA) and nifH phylogenies were congruent, while the nodA phylogeny grouped the Mesorhizobium spp. isolates with R. leguminosarum. Medicago sativa was the most sampled plant species, and only S. meliloti and R. leguminosarum were found in its nodules, while Medicago falcata nodules hosted S. meliloti and Mesorhizobium spp. Medicago lupulina was the only species that hosted all four identified rhizobial groups, including S. medicae. This study provides the first report on the Mesorhizobium spp. associated with M. falcata nodules. Additionally, R. leguminosarum and two Mesorhizobium genospecies were identified as novel symbionts for Medicago spp. Comparative analysis of Medicago-associated rhizobia from other studies revealed that differences in 16S rDNA sequence type composition were influenced by Medicago species identity rather than geographic region. Full article

Biological nitrogen fixation in legume plants depends on the diversity of rhizobia present in the soil. Rhizobial strains exhibit specificity towards host plants and vary in their capacity to fix nitrogen. The increasing interest in rhizobia diversity has prompted studies of their phylogenetic relations. Molecular identification of Rhizobium is quite complex, requiring multiple gene markers to be analysed to distinguish strains at the species level or to predict their host plant. In this research, 50 rhizobia isolates were obtained from the root nodules of five different Pisum sativum L. genotypes (“Bagoo”, “Respect”, “Astronaute”, “Lina DS”, and “Egle DS”). All genotypes were growing in the same field, where ecological farming practices were applied, and no commercial rhizobia inoculants were used. The influence of rhizobial isolates on pea root nodulation and dry biomass accumulation was determined. 16S rRNA gene, two housekeeping genes recA and atpD, and symbiotic gene nodC were analysed to characterize rhizobia population. The phylogenetic analysis of 16S rRNA gene sequences showed that 46 isolates were linked to Rhizobium leguminosarum; species complex 1 isolate was identified as Rhizobium nepotum, and the remaining 3 isolates belonged to Rahnella spp., Paenarthrobacter spp., and Peribacillus spp. genera. RecA and atpD gene analysis showed that the 46 isolates identified as R. leguminosarum clustered into three genospecies groups (B), (E) and (K). Isolates that had the highest influence on plant dry biomass accumulation clustered into the (B) group. NodC gene phylogenetic analysis clustered 46 R. leguminosarum isolates into 10 groups, and all isolates were assigned to the R. leguminosarum sv. viciae. Full article

Aeschynomene indica rhizobia (AIRs) are special classes of bacteria capable of nodulating without nodulation factors and have photosynthetic capacity. With an aim to characterize the structural variations in Bradyrhizobium genomes during its evolution, the genomes of AIRs and the reference Bradyrhizobium strains were compared utilizing molecular biology, bioinformatics, and biochemistry techniques. The presence of symbiotic nitrogen fixation (nif) genes and photosynthetic genes, as well as components of the T3SS (Type III secretion system) and T3CP (Type III chaperone) in the genome of AIRs, was also assessed. Additionally, the origin, evolutionary history, and genes associated with the NF-independent nodulation pattern in AIRs were explored. The results indicate that horizontal gene transfer events have occurred in AIRs, and three distinct origins of AIRs were estimated: early differentiated AIRs, non-symbiotic Bradyrhizobium, and non-AIRs. In contrast to the significant genetic transformations observed in the second and third groups, the first group of AIRs displays a rich evolutionary history, exhibits high species diversity, and primarily relies on vertical transmission of nitrogen fixation and photosynthetic genes. Overall, the findings provide a fundamental theoretical foundation for gaining a deeper understanding of the phylogeny and genealogy of AIRs. Full article

The rhizobacterial strain BJ3 showed 16S rDNA sequence similarity to species within the Burkholderia genus. Its complete genome sequence revealed a 97% match with Burkholderia contaminans and uncovered gene clusters essential for plant-growth-promoting traits (PGPTs). These clusters include genes responsible for producing indole acetic acid (IAA), osmolytes, non-ribosomal peptides (NRPS), volatile organic compounds (VOCs), siderophores, lipopolysaccharides, hydrolytic enzymes, and spermidine. Additionally, the genome contains genes for nitrogen fixation and phosphate solubilization, as well as a gene encoding 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The treatment with BJ3 enhanced root architecture, boosted vegetative growth, and accelerated early flowering in Arabidopsis. Treated seedlings also showed increased lignin production and antioxidant capabilities, as well as notably increased tolerance to water deficit and high salinity. An RNA-seq transcriptome analysis indicated that BJ3 treatment significantly activated genes related to immunity induction, hormone signaling, and vegetative growth. It specifically activated genes involved in the production of auxin, ethylene, and salicylic acid (SA), as well as genes involved in the synthesis of defense compounds like glucosinolates, camalexin, and terpenoids. The expression of AP2/ERF transcription factors was markedly increased. These findings highlight BJ3’s potential to produce various bioactive metabolites and its ability to activate auxin, ethylene, and SA signaling in Arabidopsis, positioning it as a new Burkholderia strain that could significantly improve plant growth, stress resilience, and immune function. Full article

Strain Q11^T of an irregular coccoid Gram-positive bacterium, aerobic and non-motile, was isolated from Meconopsis integrifolia seeds. Strain Q11^T grew optimally in 1% (w/v) NaCl, pH 7, at 30 °C. Strain Q11^T is most closely related to Flexivirga, as evidenced by 16S rRNA gene analysis, and shares the highest similarity with Flexivirga aerilata ID2601S^T (99.24%). Based on genome sequence analysis, the average nucleotide identity and digital DNA–DNA hybridization values of strains Q11^T and D2601S^T were 88.82% and 36.20%, respectively. Additionally, strain Q11^T showed the abilities of nitrogen fixation and indole acetic acid production and was shown to promote maize growth under laboratory conditions. Its genome contains antibiotic resistance genes (the vanY gene in the vanB cluster and the vanW gene in the vanI cluster) and extreme environment tolerance genes (ectoine biosynthetic gene cluster). Shotgun proteomics also detected antibiotic resistance proteins (class A beta-lactamases, D-alanine ligase family proteins) and proteins that improve plant cold tolerance (multispecies cold shock proteins). Strain Q11^T was determined to be a novel species of the genus Flexivirga, for which the name Flexivirga meconopsidis sp. nov. is proposed. The strain type is Q11^T (GDMCC 1.3002^T = JCM 36020 ^T). Full article

Fe-S clusters are essential cofactors for the activity of a large variety of metalloproteins that play important roles in respiration, photosynthesis, nitrogen fixation, regulation of gene expression, and numerous metabolic pathways, including biosynthesis of other protein cofactors. Assembly of iron and sulfur atoms into a cluster, followed by its insertion into the polypeptide chain, is a complex process ensured by multiproteic systems. Through evolution, eukaryotes have acquired two Fe-S protein biogenesis systems by endosymbiosis from bacteria. These systems, ISC and SUF, are compartmentalized in mitochondria and plastids, respectively. The eukaryotic Fe-S protein biogenesis system (CIA) is dedicated to the biogenesis of cytosolic and nuclear Fe-S proteins. While the CIA system is absent in bacteria, at least two of its components share homologies with bacterial Fe-S protein biogenesis factors, Mrp and SufT. Here, we provide an overview of the role of Mrp and SufT in Fe-S protein biogenesis in bacteria, aiming to put forward specific but also common features with their eukaryotic CIA counterparts. Full article

This study examined the biological activity and genome of Bacillus cereus CDHWZ7 isolated from the root of Lycium ruthenicum in the Dachaidan saline area, Haixi Prefecture, Qinghai Province, China. The results revealed that B. cereus CDHWZ7 exhibited strong inhibition activity against the pathogenic fungi Fusarium graminearum, F. acuminatum, and F. oxysporum. CDHWZ7 also demonstrated cellulose-degrading activity, nitrogen-fixing activity, and the ability to secrete indole-3-acetic acid (IAA) at 55.00 mg∙L⁻¹. The strain CDHWZ7 can grow at a salt concentration of 3–11%, a pH range of 5–11, and a temperature of 4 °C–18 °C, and shows good salt tolerance, acid and alkaline tolerance, and low-temperature fitness. The genome of strain CDHWZ7 was sequenced using Illumina HiSeq + PacBio, revealing a circular structure of 5,648,783 bp in length, containing two intact plasmids with an average GC content of 35.2%, and a total number of 5672 encoded genes. It contained 106 tRNA genes, 42 rRNA genes, and 134 sRNA genes. A total of 137 genes were annotated as carbohydrases, with a total base length of 3,968,396,297 bp. The numbers of coding sequences assigned to the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of Proteins, and Gene Ontology Databases were 4038, 4133, and 2160, respectively. Further analysis of the genome identified genes encoding chitinase activity, cellulases, secondary metabolites, phytohormone production, volatile compounds, nitrogen and phosphate metabolism, and resistance responses to biotic stresses (glycine betaine transporter protein, catalase, superoxide dismutase, low-affinity potassium transporter protein, cold-shock protein, heat-shock protein), as well as genes related to proliferation, stress response, and resistance to pathogenic fungi. Therefore, this study determined that strain CDHWZ7 has several excellent biological traits, such as antagonism to pathogenic fungi, nitrogen-fixation ability, cellulose-degradation ability, and IAA-production ability. The genome sequence of strain CDHWZ7 and several biodefense functional genes were also analyzed, revealing the potential use of strain CDHWZ7 in the development of biological agents. Full article

In Uruguayan soils, populations of native and naturalized rhizobia nodulate white clover. These populations include efficient rhizobia but also parasitic strains, which compete for nodule occupancy and hinder optimal nitrogen fixation by the grassland. Nodulation competitiveness assays using gusA-tagged strains proved a high nodule occupancy by the inoculant strain U204, but this was lower than the strains with intermediate efficiencies, U268 and U1116. Clover biomass production only decreased when the parasitic strain UP3 was in a 99:1 ratio with U204, but not when UP3 was at equal or lower numbers than U204. Based on phylogenetic analyses, strains with different efficiencies did not cluster together, and U1116 grouped with the parasitic strains. Our results suggest symbiotic gene transfer from an effective strain to U1116, thereby improving its symbiotic efficiency. Genome sequencing of U268 and U204 strains allowed us to assign them to species Rhizobium redzepovicii, the first report of this species nodulating clover, and Rhizobium leguminosarun, respectively. We also report the presence of hrrP- and sapA-like genes in the genomes of WSM597, U204, and U268 strains, which are related to symbiotic efficiency in rhizobia. Interestingly, we report here chromosomally located hrrP-like genes. Full article

Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N₂) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the “nitrogen problem”. Azotobacter vinelandii is a well–established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation. Full article

The plant microbiome can be used to bolster plant defense against abiotic and biotic stresses. Some strains of endophytes, the microorganisms within plants, can directly inhibit the growth of plant fungal pathogens. A previously isolated endophyte from wild Populus (poplar), WPB of the species Burkholderia vietnamiensis, had robust in vitro antifungal activity against pathogen strains that are highly virulent and of concern to Pacific Northwest agriculture: Rhizoctonia solani AG-8, Fusarium culmorum 70110023, and Gaemannomyces graminis var. tritici (Ggt) ARS-A1, as well as activity against the oomycete, Pythium ultimum 217. A direct screening method was developed for isolation of additional anti-fungal endophytes from wild poplar extracts. By challenging pathogens directly with dilute extracts, eleven isolates were found to be inhibitory to at least two plant pathogen strains and were therefore chosen for further characterization. Genomic analysis was conducted to determine if these endophyte strains harbored genes known to be involved in antimicrobial activities. The newly isolated Bacillus strains had gene clusters for production of bacillomycin, fengicyn, and bacillibactin, while the gene cluster for the synthesis of sessilin, viscosin and tolaasin were found in the Pseudomonas strains. The biosynthesis gene cluster for occidiofungin (ocf) was present in the Burkholderia vietnamiensis WPB genome, and an ocf deletion mutant lost inhibitory activity against 3 of the 4 pathogens. The new isolates lacked the gene cluster for occidiofungin implying they employ different modes of action. Other symbiotic traits including nitrogen fixation, phosphate solubilization, and the production of auxins and siderophores were investigated. Although it will be necessary to conduct in vivo tests of the candidates with pathogen-infected agricultural crops, the wild poplar tree microbiome may be a rich source of beneficial endophyte strains with potential for biocontrol applications against a variety of pathogens and utilizing varying modes of action. Full article