Nif Genes Research Articles

Construction of highly-effective symbiotic bacteria: Evolutionary models and genetic approaches

Using the example of N2-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve the following: 1) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; 2) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes, encoding for nitrogenase synthesis or for the energy supply of N2 fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars, and the production of polysaccharides, and storage compounds, as well as for oxidative-reductive processes).

The genome of Paenibacillus sabinae T27 provides insight into evolution, organization and functional elucidation of nif and nif-like genes.

BackgroundMost biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. This enzyme is a complex which contains the MoFe protein encoded by nifDK and the Fe protein encoded by nifH. In addition to nifHDK, nifHDK-like genes were found in some Archaea and Firmicutes, but their function is unclear.ResultsWe sequenced the genome of Paenibacillus sabinae T27. A total of 4,793 open reading frames were predicted from its 5.27 Mb genome. The genome of P. sabinae T27 contains fifteen nitrogen fixation (nif) genes, including three nifH, one nifD, one nifK, four nifB, two nifE, two nifN, one nifX and one nifV. Of the 15 nif genes, eight nif genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) and two non-nif genes (orf1 and hesA) form a complete nif gene cluster. In addition to the nif genes, there are nitrogenase-like genes, including two nifH-like genes and five pairs of nifDK-like genes. IS elements on the flanking regions of nif and nif-like genes imply that these genes might have been obtained by horizontal gene transfer. Phylogenies of the concatenated 8 nif gene (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) products suggest that P. sabinae T27 is closely related to Frankia. RT-PCR analysis showed that the complete nif gene cluster is organized as an operon. We demonstrated that the complete nif gene cluster under the control of σ70-dependent promoter enabled Escherichia coli JM109 to fix nitrogen. Also, here for the first time we demonstrated that unlike nif genes, the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen, and nifH-like or nifD-like gene could not restore the nitrogenase activity of Klebsiella pneumonia nifH− and nifD− mutant strains, respectively, suggesting that nifHDK-like genes were not involved in nitrogen fixation.ConclusionsOur data and analysis reveal the contents and distribution of nif and nif-like genes and contribute to the study of evolutionary history of nitrogen fixation in Paenibacillus. For the first time we demonstrated that the transcriptions of nifHDK-like genes were not regulated by ammonium and oxygen and nifHDK-like genes were not involved in nitrogen fixation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-723) contains supplementary material, which is available to authorized users.

Development of a whole community genome amplification-assisted DNA microarray method to detect functional genes involved in the nitrogen cycle.

A novel DNA microarray analysis targeting key functional genes involved in most nitrogen cycling reactions was developed to comprehensively analyze microbial populations associated with the nitrogen cycle. The developed microarray contained 876 oligonucleotide probes based on the nucleotide sequences of the nif, amo, hao/hzo, nap, nar, nirK, nirS, nrf, cnor, qnor and nos genes. An analytical method combining detection by the designed microarray with whole community genome amplification was then applied to monitor the nitrogen cycling microorganisms in river water and wastewater treatment sludge samples. The developed method revealed that nitrogen cycling microorganisms in river water appeared to become less diverse in response to input of effluent from municipal wastewater treatment plants. Additionally, the nitrogen cycling community associated with anaerobic ammonium oxidation and partial nitrification reactors could be reasonably analyzed by the developed method. However, the results obtained for two activated sludge samples from municipal wastewater treatment plants with almost equivalent wastewater treatment performance differed greatly from each other. These results suggested that the developed method is useful for comprehensive analysis of nitrogen cycling microorganisms, although its applicability to complex samples with abundant untargeted populations should be further examined.

Borrelia spirochetes in Russia: Genospecies differentiation by real-time PCR

Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis which is widespread in Russia. Nowadays, three clinically important B. burgdorferi s.l. genospecies, B. afzelii, B. garinii, B. bavariensis sp. nov., can be found in Russia, as well as B. miyamotoi, which belongs to the tick-borne relapsing fever group of spirochetes. Several techniques have been developed to differentiate Borrelia genospecies. However, most of them do not allow detection of all of these genospecies simultaneously. Also, no method based on the RT-PCR TaqMan approach has been proposed to differentiate the genetically closely related species B. bavariensis and B. garinii. In the present paper, we investigated two species of ticks, I. persulcatus and I. pavlovskyi (1343 and 92 adults, respectively). Two sets of primers and probes for RT-PCR, with uvrA, glpQ and nifS genes as targets, were designed to detect four Borrelia genospecies in positive samples. The average prevalence of Borrelia sp. was about 40%, with B. afzelii as the most prevalent genospecies. Mixed infections of B. bavariensis and B. garinii were found to be extremely rare. While B. bavariensis was predominant in I. persulcatus, I. pavlovskyi ticks were infected exclusively by B. garinii. The proposed technique proved to be efficient in selection of individual Borrelia species for further genetic analysis, in particular, for multilocus sequence typing. Also, it could be applied for the differentiation of Borrelia genospecies in clinical material.

The transcriptional activator NrpA is crucial for inducing nitrogen fixation in Methanosarcina mazeiGö1 under nitrogen-limited conditions.

With the aim of unraveling their potential involvement in the regulation of nitrogen metabolism in Methanosarcina mazei strain Gö1, we characterized five genes that are differentially transcribed in response to changing nitrogen availability and encoding putative transcriptional regulators. Study of the respective mutant strains under nitrogen-limited conditions revealed a growth delay for M. mazei MM0444::pac and MM1708::pac, and strongly reduced diazotrophic growth for MM0872::pac, whereas the absence of MM2441 or MM2525 did not affect growth behaviour. Transcriptome analyses further demonstrated that only MM1708 - encoding a CxxCG zinc finger protein - plays a regulatory role in nitrogen metabolism, most likely by specifically enhancing transcription of the N2 fixation (nif) operon under nitrogen-limited conditions. In agreement with this, a palindromic binding motif was predicted in silico in the nifH promoter region, nine nucleotides upstream of the BRE box, and confirmed to bind purified maltose-binding protein-MM1708 by electromobility shift assays. As MM1708 itself is under the control of the global nitrogen repressor NrpR, this adds a secondary level to the transcriptional regulation of the nif genes, and is most likely crucial for maximal nif induction under nitrogen-limited conditions. This is in accordance with the finding that protein expression of NifH is highly reduced in the absence of MM1708 under nitrogen-limited conditions. On the basis of our findings, we hypothesize that, in M. mazei, nitrogen fixation is controlled by a hierarchical network of two transcriptional regulators, the global nitrogen repressor NrpR, and the newly identified activator NrpA (MM1708), thereby providing tight control of N2 fixation.

Impact of Pesticides on PGPR Activity of Azotobacter sp. Isolated from Pesticide Flooded Paddy Soils

Azotobacter strains were isolated from paddy soils by serial dilution agar plate method. The colonies were glistening; smooth, slimy, brown to black in morphology on Jenson’s N-free agar plates and the cells were Gram- negative bacteria. Biochemically, they were positive for indole production, citrate, catalase and Voges Proskauer test. Further, Nif gene sequence results revealed the presence of seven Azotobacter species. The effect of 1, 3 and 5% pesticides concentration viz., pendimethalin, chloropyrifos, glyphosate and phorate on nitrogen fixation, indole acetic acid, gibberllic acid, phosphate solubilization and bioassay of Azotobacter sp. were studied. Among all the species, GVT-1 strain was found to fix nitrogen at a maximum of 30µgN2 ml-1 day-1, produced highest quantity gibberllic acid (10µg25ml-1) and able to solubilize the phosphate at the rate of 9.8cm by forming the halo zone which was supplemented with 5% phorate. Similarly, GVT-1 strain produced a highest amount of indole acetic acid (31.8µgml-1) in 5% pendimethalin. Further bioassay activities of GVT-1 strain found efficient in increasing the root, shoot length and vigor of the plant. From these results it is clear that the Azotobacter sp. not only produces plant growth promoting substances but is also resistant to different pesticides and is not affected by the bacterial activity.

Classification and phylogeny of the cyanobiont Anabaena azollae Strasburger: an answered question?

The symbiosis Azolla-Anabaena azollae, with a worldwide distribution in pantropical and temperate regions, is one of the most studied, because of its potential application as a biofertilizer, especially in rice fields, but also as an animal food and in phytoremediation. The cyanobiont is a filamentous, heterocystic cyanobacterium that inhabits the foliar cavities of the pteridophyte and the indusium on the megasporocarp (female reproductive structure). The classification and phylogeny of the cyanobiont is very controversial: from its morphology, it has been named Nostoc azollae, Anabaena azollae, Anabaena variabilis status azollae and recently Trichormus azollae, but, from its 16S rRNA gene sequence, it has been assigned to Nostoc and/or Anabaena, and from its phycocyanin gene sequence, it has been assigned as non-Nostoc and non-Anabaena. The literature also points to a possible co-evolution between the cyanobiont and the Azolla host, since dendrograms and phylogenetic trees of fatty acids, short tandemly repeated repetitive (STRR) analysis and restriction fragment length polymorphism (RFLP) analysis of nif genes and the 16S rRNA gene give a two-cluster association that matches the two-section ranking of the host (Azolla). Another controversy surrounds the possible existence of more than one genus or more than one species strain. The use of freshly isolated or cultured cyanobionts is an additional problem, since their morphology and protein profiles are different. This review gives an overview of how morphological, chemical and genetic analyses influence the classification and phylogeny of the cyanobiont and future research.

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N2-fixing strains. The heterogeneity of the 31 genomes (15 N2-fixing and 16 non-N2-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N2-fixing strains. The nif cluster is under control of a σ70-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N2-fixing and non-N2-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N2-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N2-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N2-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.

Metabolic engineering of Rhodobacter sphaeroides for improved hydrogen production

Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic purple nonsulfur bacterium that can generate hydrogen by using sunlight and organic carbon sources under anaerobic conditions. Hydrogen is produced by the nitrogenase. To optimize hydrogen production, we combined in a single strain several mutations known to enhance hydrogen production in anoxygenic phototrophic bacteria. The first metabolic engineering approach involved elimination of the major reductive pathways (electron sinks) that compete for electrons with the nitrogenase. A strain containing mutations in the pathways involving hydrogen uptake hydrogenase (hupSL), poly-β-hydroxybutyrate synthesis (phbC), and light harvesting complex-II (pucBA) showed significantly increased hydrogen production. The second approach involved increasing immediate electron flow to nitrogenase via the Rnf complex. This strategy resulted in modest improvements in hydrogen yield. The third approach involved upregulating nitrogenase expression and decoupling its expression from the presence of ammonium in the growth media. Mutations in the nifA gene, encoding the master transcriptional activator of nitrogen fixation genes, were found to be most effective for increasing total hydrogen yield, accelerating hydrogen production, and making this process tolerant to the presence of ammonia in the medium. The combination of beneficial mutations resulted in an additive and superior hydrogen producing strain of R. sphaeroides.

Transcriptional profiling of nitrogen fixation and the role of NifA in the diazotrophic endophyte Azoarcus sp. strain BH72.

BackgroundThe model endophyte Azoarcus sp. strain BH72 is known to contribute fixed nitrogen to its host Kallar grass and also expresses nitrogenase genes endophytically in rice seedlings. Availability of nitrogen is a signal regulating the transcription of nitrogenase genes. Therefore, we analysed global transcription in response to differences in the nitrogen source.Methodology/Principal FindingsA DNA microarray, comprising 70-mer oligonucleotides representing 3989 open reading frames of the genome of strain BH72, was used for transcriptome studies. Transcription profiles of cells grown microaerobically on N2 versus ammonium were compared. Expression of 7.2% of the genes was significantly up-regulated, and 5.8% down-regulated upon N2 fixation, respectively. A parallel genome-wide prediction of σ54-type promoter elements mapped to the upstream region of 38 sequences of which 36 were modulated under the N2 response. In addition to modulation of genes related to N2 fixation, the expressions of gene clusters that might be related to plant-microbe interaction and of several transcription factors were significantly enhanced. While comparing under N2-fixation conditions the transcriptome of wild type with a nifLA− insertion mutant, NifA being the essential transcriptional activator for nif genes, 24.5% of the genome was found to be affected in expression. A genome-wide prediction of 29 NifA binding sequences matched to 25 of the target genes whose expression was differential during microarray analysis, some of which were putatively negatively regulated by NifA. For selected genes, differential expression was corroborated by real time RT-PCR studies.Conclusion/SignificanceOur data suggest that life under conditions of nitrogen fixation is an important part of the lifestyle of strain BH72 in roots, as a wide range of genes far beyond the nif regulon is modulated. Moreover, the NifA regulon in strain BH72 appears to encompass a wider range of cellular functions beyond the regulation of nif genes.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Phenotypic and molecular characterization of native Azospirillum strains from rice fields to improve crop productivity

Beneficial microorganisms have been considered as an important tool for crop improvement. Native isolates of Azospirillum spp. were obtained from the rhizospheres of different rice fields. Phenotypic, biochemical and molecular characterizations of these isolates led to the identification of six efficient strain of Azospirillum. PCR amplification of the nif genes (nifH, nifD and nifK) and protein profile of Azospirillum strains revealed inter-generic and inter-specific diversity among the strains. In vitro nitrogen fixation performance and the plant growth promotion activities, viz. siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH3, phosphorus metabolism, ACC deaminase and iron tolerance were found to vary among the Azospirillum strains. The effect of Azospirillum formulations on growth of rice var. Khandagiri under field condition was evaluated, which revealed that the native formulation of Azospirillum of CRRI field (As6) was most effective to elevate endogenous nutrient content, and improved growth and better yield are the result. The 16S rRNA sequence revealed novelty of native Azospirillum lipoferum (As6) (JQ796078) in the NCBI database.

Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

BackgroundRhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids.ResultsGenome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones.ConclusionVariable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

Characterization of Klebsiella sp. Strain 10982, a Colonizer of Humans That Contains Novel Antibiotic Resistance Alleles and Exhibits Genetic Similarities to Plant and Clinical Klebsiella Isolates

A unique Klebsiella species strain, 10982, was cultured from a perianal swab specimen obtained from a patient in the University of Maryland Medical Center intensive care unit. Klebsiella sp. 10982 possesses a large IncA/C multidrug resistance plasmid encoding a novel FOX AmpC β-lactamase designated FOX-10. A novel variant of the LEN β-lactamase was also identified. Genome sequencing and bioinformatic analysis demonstrated that this isolate contains genes associated with nitrogen fixation, allantoin metabolism, and citrate fermentation. These three gene regions are typically present in either Klebsiella pneumoniae clinical isolates or Klebsiella nitrogen-fixing endophytes but usually not in the same organism. Phylogenomic analysis of Klebsiella sp. 10982 and sequenced Klebsiella genomes demonstrated that Klebsiella sp. 10982 is present on a branch that is located intermediate between the genomes of nitrogen-fixing endophytes and K. pneumoniae clinical isolates. Metabolic features identified in the genome of Klebsiella sp. 10982 distinguish this isolate from other Klebsiella clinical isolates. These features include the nitrogen fixation (nif) gene cluster, which is typically present in endophytic Klebsiella isolates and is absent from Klebsiella clinical isolates. Additionally, the Klebsiella sp. 10982 genome contains genes associated with allantoin metabolism, which have been detected primarily in K. pneumoniae isolates from liver abscesses. Comparative genomic analysis of Klebsiella sp. 10982 demonstrated that this organism has acquired genes conferring new metabolic strategies and novel antibiotic resistance alleles, both of which may enhance its ability to colonize the human body.

Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

Nitrogen fixation is a tightly regulated trait. Switching from N2 fixation-repressing conditions to the N2-fixing state is carefully controlled in diazotrophic bacteria mainly because of the high energy demand that it imposes. By using quantitative real-time PCR and quantitative immunoblotting, we show here how nitrogen fixation (nif) gene expression develops in Azotobacter vinelandii upon derepression. Transient expression of the transcriptional activator-encoding gene, nifA, was followed by subsequent, longer-duration waves of expression of the nitrogenase biosynthetic and structural genes. Importantly, expression timing, expression levels, and NifA dependence varied greatly among the nif operons. Moreover, the exact concentrations of Nif proteins and their changes over time were determined for the first time. Nif protein concentrations were exquisitely balanced, with FeMo cofactor biosynthetic proteins accumulating at levels 50- to 100-fold lower than those of the structural proteins. Mutants lacking nitrogenase structural genes or impaired in FeMo cofactor biosynthesis showed overenhanced responses to derepression that were proportional to the degree of nitrogenase activity impairment, consistent with the existence of at least two negative-feedback regulatory mechanisms. The first such mechanism responded to the levels of fixed nitrogen, whereas the second mechanism appeared to respond to the levels of the mature NifDK component. Altogether, these findings provide a framework to engineer N2 fixation in nondiazotrophs.

Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been recommended for use in commercial inoculants in Brazil. Effective methods are necessary to monitor PGPB strains in the rhizosphere. Our purpose was to develop a real time PCR method for detection of A. brasilense strain FP2 in maize seedlings targeting nifA. Primer pairs were designed and their specificity was verified using DNA from 12 different bacterial species. Standard curves were prepared for DNA quantification using serial dilution of A. brasilense DNA extracts. PCR efficiencies and correlation coefficient presented values within the acceptable range for qPCR, mean PCR efficiency was 95 % and correlation coefficient was 0.98, indicating that nifA gene was suitable for the quantitative analysis of the target bacterial genome. Inoculated maize seedlings were grown in vitro or in pots, bacterial DNA copy number per gram of fresh root was quantified 1, 4, 7 and 10 days after inoculation. The developed primers targeting nifA will be useful for monitoring Azospirillum brasilense strain FP2 in crops.

A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

English

This study was designed to evaluate the effects of three pesticides (Imazetapir, Dimethoate and Bayleton 50) at the recommended concentration (in the field), on the growth of pure cultures ofAzotobacter chroococcum and Azotobacter vinelandii, on the amount of fixed nitrogen and nifgenes. Herbicide Imazetapir had no negative effect on nitrogen fixing bacteria, while Dimethoate and Bayleton 50 exhibited inhibitory effect on growth. Same effects were obtained on fixed nitrogen obtained when treated with studied pesticides. nifH1, nifH2, nifH3, nifU and nifV from A. chroococcum, and nifH, nifK, nifD, and nifM gene in A. Vinelandii were lost when pots were cultivated with wheat and treated with both Dimethoate and Bayleton 50, therefore, be deemed highly susceptible to them. While herbicide did not affect the nif genes, the bands on gel electrophoresis appeared as normal sample.   Key words: Azotobacter chroococcum, Azotobacter vinelandii, fungicide (Bayleton 50), herbicide (Imazetapir), insecticide (Dimethoate).

Using Synthetic Biology to Distinguish and Overcome Regulatory and Functional Barriers Related to Nitrogen Fixation

Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ∼100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase–LacI expression system was used to replace the σ54-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ∼42% of the nitrogenase activity of the σ54-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.

In-silico analysis suggests alterations in the function of XisA protein as a possible mechanism of butachlor toxicity in the nitrogen fixing cyanobacterium Anabaena sp. PCC 7120.

Butachlor, a commonly used herbicide adversely affects the nitrogen fixing capability of Anabaena, an acclaimed nitrogen fixer in the Indian paddy fields. The nitrogen fixation in Anabaena is triggered by the excision of nifD element by xisA gene leading to rearrangement of nifD forming nifHDK operon in the heterocyst of Anabaena sp. PCC7120. Functional elucidation adjudged through in-silico analysis revealed that xisA belongs to integrase family of tyrosine recombinase. The predicted functional partners with XisA protein that have shown cooccurence with this protein in a network are mainly hypothetical proteins with unknown functions except psaK1 whose exact function in photosystem I is not yet known. The focus of this study was to find out the relation between XisA and butachlor using in-silico approaches. The XisA protein was modeled and its active sites were identified. Docking studies revealed that butachlor binds at the active site of XisA protein hampering its excision ability vis-à-vis nif genes in Anabaena sp. PCC7120. This study reveals that butachlor is not directly involved in hampering the nitrogen fixing ability of Anabaena sp. PCC7120 but by arresting the excision ability of XisA protein necessary for the functioning of nif gene and nitrogen fixation.