Nif Genes Research Articles

Revealing the underlying mechanisms mediated by endophytic actinobacteria to enhance the rhizobia - chickpea (Cicer arietinum L.) symbiosis

PurposeThe effects of endophytic actinobacterial strains, Microbispora sp. CP56, Actinomadura sp. CP84B, Streptomyces spp. CP200B and CP21A, on the chickpea-Mesorhizobium symbiosis, were investigated in planta, with the aim of revealing the underlying mechanisms of action.MethodsThe actinobacterial endophytes were co-inoculated with Mesorhizobium ciceri onto chickpea seedlings to study the effect on plant growth parameters, nodulation development and grain yield. The role of actinobacterial exudates on rhizobial growth was investigated, as was the role of root exudates of actinobacteria-colonized plants on the expression of rhizobial nod factors and biofilm formation. Changes in expression of plant flavonoids and bacterial N-fixation genes resulting from actinobacterial co-inoculation were assessed using qPCR.ResultsApplication of actinobacterial endophytes, together with M. ciceri, showed growth promotion of chickpea with an increase in root nodule number and weight. Enhanced nodulation was accompanied by increases in total plant nitrogen, larger total plant weight and a 2–3-fold increase in grain yield. Factors associated with this tripartite symbiosis are promotion of rhizobial growth, earlier nodule formation, increased secondary root formation, up-regulated expression of genes related to flavonoid synthesis and nif genes. In addition, exudates of chickpea roots colonised with actinobacteria increased nodulation-related biological processes - rhizobial chemotaxis, biofilm formation and nod gene expression.ConclusionThese endophytic actinobacteria positively affect many aspects of the chickpea-Mesorhizobium symbiosis resulting in increases in grain yield. Similar improvements recorded in chickpea growing in potted field soils, shows the potential to enhance chickpea production in the field.

Genomic insights into the plant-associated lifestyle of Kosakonia radicincitans MUSA4, a diazotrophic plant-growth-promoting bacterium

The process of nitrogen (N) fixation by plant-associated bacteria plays an indispensable role in the development of novel agricultural solutions worldwide. In this sense, it is of extreme importance to identify and understand the properties of efficient plant-growth-promoting bacteria (PGPB) that are able to fix N. In this study, the characterization and detailed genomic analysis of the diazotrophic bacterium Kosakonia radicincitans MUSA4, isolated from the internal leaf tissues of a banana tree in Brazil, were undertaken.K. radicincitans MUSA4 presented several plant-growth-promoting traits, including indoleacetic acid, siderophore, acetoin and polyamine biosynthesis, phosphate solubilization, and nitrogen fixation. The strain was able to increase cucumber plant growth significantly, demonstrating its potential in beneficial interactions with plant hosts. Detailed genomic analysis of strain MUSA4 revealed the abundant presence of genes involved in plant colonization, stress resistance and plant-growth-promoting abilities. Moreover, the genome harbored the nif and anf gene clusters, encoding the Fe-Mo nitrogenase and Fe-Fe nitrogenase systems, respectively. Comparative genomic analysis also showed that strain MUSA4 possessed several strain-specific genes, which could be related to its evolutionary history in Brazilian mangrove environments.The results obtained in the present study revealed the plant beneficial role and biotechnological potential of K. radicincitans MUSA4, and provided new insights into plant colonization and plant growth promoting mechanisms employed by diazotrophic Kosakonia.

Nitrogen fixation by Paenibacillus polymyxa WLY78 is responsible for cucumber growth promotion

To study nitrogen contribution to cucumber derived from nitrogen fixation of Paenibacillus polymyxa WLY78. The nif gene cluster deletion mutant (ΔnifB-V) of Paenibacillus polymyxa WLY78 was constructed by a homologous recombination method. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen fixation contribution was estimated by 15N isotope dilution method (also being called the 15N natural abundance technique). Deletion of nif gene cluster of P. polymyxa WLY78 resulted in complete loss of nitrogenase activity. Greenhouse experiments showed that inoculation with P. polymyxa WLY78 could significantly enhance the lengths and dry weights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant could not. 15N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen fixation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly could not provide nitrogen for plant growth. This present study demonstrated that nitrogen fixation performed by P. polymyxa WLY78 contributes to plant growth.

Description of two nitrogen-fixing bacteria, Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., isolated from paddy soils.

Two strictly anaerobic nitrogen-fixing strains, designated RG17T and RG53T, were isolated from paddy soils in China. Strains RG17T and RG53T showed the highest 16S rRNA gene sequence similarities to the type strain Geomonas paludis (97.9-98.4%). Phylogenetic tree based on 16S rRNA gene sequences showed that two strains clustered with members of the genus Geomonas. Growth of strain RG17T was observed at 20-42°C, pH 5.5-8.5 and 0-0.3% (w/v) NaCl while strain RG53T growth was observed at 20-42°C, pH 5.5-9.5 and 0-0.7% (w/v) NaCl. Strains RG17T and RG53T contained MK-8 as main menaquinone and C15:1 ω6c, iso-C15:0, and Summed Feature 3 as the major fatty acids. The genomic DNA G + C content of strains RG17T and RG53T were 61.6 and 60.7%, respectively. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the isolated strains and the closely related Geomonas species were lower than the cut-off value (dDDH 70% and ANI 95-96%) for prokaryotic species delineation. Both strains possessed nif genes nifHDK and nitrogenase activities. Based on the above results, the two strains represent two novel species of the genus Geomonas, for which the names Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., are proposed. The type strains are RG17T (= GDMCC 1.2687T = KTCC 25332T) and RG53T (= GDMCC 1.2630T = KCTC 25331T), respectively.

Paenibacillus piscarius sp. nov., a novel nitrogen-fixing species isolated from the gut of the armored catfish Parotocinclus maculicauda.

A Gram-positive, nitrogen-fixing and endospore-forming strain, designated P121T, was isolated from the gut of the armored catfish (Parotocinclus maculicauda) and identified as a member of the genus Paenibacillus based on the sequences of the 16S rRNA encoding gene, rpoB, gyrB and nifH genes and phenotypic analyses. The most closely related species to strain P121T were Paenibacillus rhizoplanae DSM 103993T, Paenibacillus silagei DSM 101953T and Paenibacillus borealis DSM 13188T, with similarity values of 98.9, 98.3 and 97.6%, respectively, based on 16S rRNA gene sequences. Genome sequencing revealed a genome size of 7,513,698bp, DNA G + C content of 53.9mol% and the presence of the structural nitrogenase encoding genes (nifK, nifD and nifH) and of other nif genes necessary for nitrogen fixation. Digital DNA-DNA hybridization (dDDH) experiments and average nucleotide identity (ANI) analyses between strain P121T and the type strains of the closest species demonstrated that the highest values were below the thresholds of 70% dDDH (42.3% with P. borealis) and 95% ANI (84.28% with P. silagei) for bacterial species delineation, indicating that strain P121T represents a distinct species. Its major cellular fatty acid was anteiso-C15:0 (42.4%), and the major isoprenoid quinone was MK-7. Based on physiological, genomic, biochemical and chemotaxonomic characteristics, we propose that strain P121T represents a novel species for which the name Paenibacillus piscarius sp. nov. is proposed (type strain = DSM 25072 = LFB-Fiocruz 1636).

Detection of endophytic association between Aeschynomene nodulating Bradyrhizobium sp. and traditional Desariya rice roots under rice-Aeschynomene ecosystem of chaur land, Bihar, India

Engineering diazotrophic rice having either an integral component of diazotrophic microbes or placing microbial origin nif gene to the rice plant is the dream of biotechnologist. Rice-Aeschynomene ecosystem of pristine chaur land provides a suitable niche to search Rhizobium endophytes in rice. Accordingly, the work was initiated to search suitable endophytic Rhizobium strain for artificial symbiosis within the roots of Desariya rice and its source through morphological, biochemical and molecular approaches. Detection of Acetylene reduction assay (ARA) activity in sterilized Desariya rice root confirmed the presence of putative diazotrophic endophytes in rice root. Isolates from Aeschynomene aspera L. nodulating and Desariya rice endophytic Rhizobium were evaluated for growth, IAA, morphological and biochemical features. Carbon profiling pattern of both these isolates indicated that Desariya rice endophytic Rhizobium has its similarity with Aeschynomene aspera L. nodulating Rhizobium. 16S rRNA gene sequencing confirmed the presence of endophytic Bradyrhizobium sp. in Desariya rice roots and its similarity with Aeschynomene aspera L. nodulating Bradyrhizobium. Desariya rice Bradyrhizobium may be an ideal candidate in the future for creating artificial symbiosis in rice due to its similarity with Aeschynomene aspera L. Bradyrhizobium.

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

(Keynote) Engineering Cyanobacteria for Nitrogen Reduction

Ammonia is currently the second most widely produced chemical in the world (second to sulfuric acid). Most ammonia (over 66%) is now produced through the Haber−Bosch process, consuming approximately 5% of the global natural gas production and contributes to over 3% of global carbon dioxide emissions. In an effort to develop an electrochemical alternative, we have engineered cyanobacterium to perform nitrogen fixation under ambient conditions without consuming natural gas, in stark contrast to the Haber-Bosch process that requires extreme temperatures and pressures to the same end. To realize the heterologous expression of nitrogenase, we transformed the cyanobacterium S. elongatus PCC 7942 with the minimal nitrogen fixation (nif) gene cluster. Nitrogenase can reduce N2 to NH3 at mild conditions (< 40 oC, atmospheric pressure). There are three known types of nitrogenase, in which the central composition of their active-site metalloclusters are molybdenum(Mo), vanadium (V), and iron (Fe), respectively. Most biological nitrogen fixation is catalyzed by the Mo-based nitrogenase enzyme. Biosynthesis of Mo-nitrogenase generally requires a large number of nif genes. However, the transformation and transcription of such a larger set of gene cluster into the cyanobacterium is challenging. In this work, a minimal nif cluster with reduced genetic complexity was chosen to accomplish the transformation of the cyanobacterium S. elongatus PCC 7942. The cyanobacterium S. elongatus PCC 7942 was transformed with nif genes and it was shown to be able to express active nitrogenase. The engineered S. elongatus PCC 7942 was immobilized on an electrode to accelerate the microbial conversion of nitrogen into ammonium using electrochemical driving forces. Methyl viologen (MV) was added as an electron mediator to shuttle electrons from the electrode to the enzyme active center, since MV can go through the cell membrane and deliver continuous electrons to nitrogenase.

Azospirillum brasilense Az39 restricts cadmium entrance into wheat plants and mitigates cadmium stress

In this work, we tested if inoculation with the plant growth-promoting rhizobacteria Azospirillum brasilense strain Az39 alleviates Cd2+ stress in wheat seedlings grown under controlled conditions. Growth, total N, N-related metabolites/enzymes, and oxidative stress parameters were measured. Additionally, the usefulness of a real-time PCR protocol to screen the preferred colonization site of the introduced microorganism was evaluated. Inoculated plants demonstrated mitigation of cadmium-induced adverse effects on plant growth and less reactive oxygen species accumulation in their roots by the end of the experiment, 28 days after sowing. Cd addition resulted in lower NO3− content in the leaves and higher NO3− content in the roots, and a significant rise in NH4+ concentration in both organs in uninoculated plants; in inoculated plants, NH4+ content in the roots did not vary. A. brasilense Az39 enhanced NO levels in wheat root tips, and more adventitious roots and root hairs were observed in inoculated plants. Despite having a more developed root system, inoculated plants showed lower Cd levels in their roots compared to non-inoculated plants. Inoculation with this PGPR favored ion homeostasis in the roots of metal-exposed plants, decreasing Cd/Fe ratio. We corroborated A. brasilense Az39 preference for wheat exorhizosphere using a real-time PCR-based method targeting the nifA gene.

Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1–1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1–1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

Phylogeny of Nitrogenase Structural and Assembly Components Reveals New Insights into the Origin and Distribution of Nitrogen Fixation across Bacteria and Archaea.

The phylogeny of nitrogenase has only been analyzed using the structural proteins NifHDK. As nifHDKENB has been established as the minimum number of genes necessary for in silico prediction of diazotrophy, we present an updated phylogeny of diazotrophs using both structural (NifHDK) and cofactor assembly proteins (NifENB). Annotated Nif sequences were obtained from InterPro from 963 culture-derived genomes. Nif sequences were aligned individually and concatenated to form one NifHDKENB sequence. Phylogenies obtained using PhyML, FastTree, RapidNJ, and ASTRAL from individuals and concatenated protein sequences were compared and analyzed. All six genes were found across the Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota. The phylogenies of individual Nif proteins were very similar to the overall NifHDKENB phylogeny, indicating the assembly proteins have evolved together. Our higher resolution database upheld the three cluster phylogeny, but revealed undocumented horizontal gene transfers across phyla. Only 48% of the 325 genera containing all six nif genes are currently supported by biochemical evidence of diazotrophy. In addition, this work provides reference for any inter-phyla comparison of Nif sequences and a quality database of Nif proteins that can be used for identifying new Nif sequences.

Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation.

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

Functional analysis of multiple nifB genes of Paenibacillus strains in synthesis of Mo-, Fe- and V-nitrogenases

BackgroundBiological nitrogen fixation is catalyzed by Mo-, V- and Fe-nitrogenases that are encoded by nif, vnf and anf genes, respectively. NifB is the key protein in synthesis of the cofactors of all nitrogenases. Most diazotrophic Paenibacillus strains have only one nifB gene located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV). But some Paenibacillus strains have multiple nifB genes and their functions are not known.ResultsA total of 138 nifB genes are found in the 116 diazotrophic Paenibacillus strains. Phylogeny analysis shows that these nifB genes fall into 4 classes: nifBI class including the genes (named as nifB1 genes) that are the first gene within the compact nif gene cluster, nifBII class including the genes (named as nifB2 genes) that are adjacent to anf or vnf genes, nifBIII class whose members are designated as nifB3 genes and nifBIV class whose members are named as nifB4 genes are scattered on genomes. Functional analysis by complementation of the ∆nifB mutant of P. polymyxa which has only one nifB gene has shown that both nifB1 and nifB2 are active in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Deletion analysis also has revealed that nifB1 of Paenibacillus sabinae T27 is involved in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Complementation of the P. polymyxa ∆nifBHDK mutant with the four reconstituted operons: nifB1anfHDGK, nifB2anfHDGK, nifB1vnfHDGK and nifB2vnfHDGK, has shown both that nifB1 and nifB2 were able to support synthesis of Fe- or V-nitrogenases. Transcriptional results obtained in the original Paenibacillus strains are consistent with the complementation results.ConclusionsThe multiple nifB genes of the diazotrophic Paenibacillus strains are divided into 4 classes. The nifB1 located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV) and the nifB2 genes being adjacent to nif or anf or vnf genes are active in synthesis of Mo-, Fe and V-nitrogenases, but nifB3 and nifB4 are not. The reconstituted anf system comprising 8 genes (nifBanfHDGK and nifXhesAnifV) and vnf system comprising 10 genes (nifBvnfHDGKEN and nifXhesAnifV) support synthesis of Fe-nitrogenase and V-nitrogenase in Paenibacillus background, respectively.

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N2 fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261–357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.

Functional Specificity of the nifA Gene Product within the Group of Root Nodule Bacteria

Functional Specificity of the nifA Gene Product within the Group of Root Nodule Bacteria

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

Disrupting hierarchical control of nitrogen fixation enables carbon-dependent regulation of ammonia excretion in soil diazotrophs.

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

Evolutionary origin and ecological implication of a unique nif island in free-living Bradyrhizobium lineages.

The alphaproteobacterial genus Bradyrhizobium has been best known as N2-fixing members that nodulate legumes, supported by the nif and nod gene clusters. Recent environmental surveys show that Bradyrhizobium represents one of the most abundant free-living bacterial lineages in the world's soils. However, our understanding of Bradyrhizobium comes largely from symbiotic members, biasing the current knowledge of their ecology and evolution. Here, we report the genomes of 88 Bradyrhizobium strains derived from diverse soil samples, including both nif-carrying and non-nif-carrying free-living (nod free) members. Phylogenomic analyses of these and 252 publicly available Bradyrhizobium genomes indicate that nif-carrying free-living members independently evolved from symbiotic ancestors (carrying both nif and nod) multiple times. Intriguingly, the nif phylogeny shows that the vast majority of nif-carrying free-living members comprise an independent cluster, indicating that horizontal gene transfer promotes nif expansion among the free-living Bradyrhizobium. Comparative genomics analysis identifies that the nif genes found in free-living Bradyrhizobium are located on a unique genomic island of ~50 kb equipped with genes potentially involved in coping with oxygen tension. We further analyze amplicon sequencing data to show that Bradyrhizobium members presumably carrying this nif island are widespread in a variety of environments. Given the dominance of Bradyrhizobium in world's soils, our findings have implications for global nitrogen cycles and agricultural research.