scholarly journals Characterisation of a Novel Nitrogen-Fixing, Polyhydroxyalkanoate-Producing Bacterium, Novosphingobium Nitrogenifigens Y88T

2021 ◽  
Author(s):  
◽  
Anne-Marie Smit
Keyword(s):  
Nitrogen Fixation ◽  
Model Organism ◽  
Dry Weight ◽  
Growth Conditions ◽  
Biofuel Production ◽  
Nitrogen Fixing ◽  
Production Environment ◽  
Amino Terminal ◽  
Nitrogen Utilisation ◽  
Pha Production

<p>The novel sphingomonad Novosphingobium nitrogenifigens Y88T (Y88T) is an obligate aerobe able to grow in nutrient-imbalanced environments where nitrogen is naturally limiting, but carbon is found in abundance. Due to its ability to fix atmospheric nitrogen and produce the bioplastic polyhydroxyalkanoate (PHA), Y88T is well-suited for growth in a nitrogenlimited but carbon-enriched environment. Because of these metabolic abilities, Y88T is of interest as a model organism for PHA production unconstrained by nitrogen-limiting conditions. Growth profiles and PHA production profiles were determined for Y88T under conditions of carbon enrichment, nitrogen sufficiency and depletion to investigate carbon and nitrogen utilisation as well as PHA production in this organism. Also, since the nitrogenase enzyme required for nitrogen fixation is oxygen labile, the effect of DO concentration and the relationship between aerobic metabolism and the nitrogen-fixing and PHA-producing abilities of Y88T was investigated. This study demonstrated: that glucose is the preferred growth substrate for Y88T; that no direct relationship exists between nitrogen fixation and PHB accumulation in Y88T; that Y88T can reliably produce in excess of 80 % of its dry weight as polyhydroxybutyrate (PHB), a type of PHA, from glucose under nitrogenlimiting conditions. Proteomic signatures were determined for the various physiological responses of Y88T to growth, nitrogen utilisation, PHB production and exposure to different levels of DO. More than 250 unique proteins, including the core nitrogen-fixation, PHB-synthetic and glycolytic proteins were identified. Y88T apparently converts glucose to PHB via three interrelated glucose catabolic pathways and proteins likely involved in these pathways were identified. This study revealed that, regardless of growth conditions and despite decreased abundance of the Y88T nitrogenase enzyme, growth and PHB synthesis were not inhibited at DOhigh concentrations. Proteomic characterisation of the Y88T phasin, a PHA granule-associated protein, iii identified an amino-terminal, low complexity alanine and proline rich segment found only in other sphingomonads. The expression level of the Y88T phasin correlated well with PHB yields, suggesting the use of this protein as a biomarker to optimise PHB yield in a production environment. Y88T has the potential to be a useful production strain in pure culture, utilising its natural and robust propensity to metabolise glucose to preferentially produce PHB. Targets for biotechnological improvement and the potential for application of Y88T to biofuel production are discussed.</p>

scholarly journals Characterisation of a Novel Nitrogen-Fixing, Polyhydroxyalkanoate-Producing Bacterium, Novosphingobium Nitrogenifigens Y88T

2021 ◽  
Author(s):  
◽  
Anne-Marie Smit
Keyword(s):  
Nitrogen Fixation ◽  
Model Organism ◽  
Dry Weight ◽  
Growth Conditions ◽  
Biofuel Production ◽  
Nitrogen Fixing ◽  
Production Environment ◽  
Amino Terminal ◽  
Nitrogen Utilisation ◽  
Pha Production

<p>The novel sphingomonad Novosphingobium nitrogenifigens Y88T (Y88T) is an obligate aerobe able to grow in nutrient-imbalanced environments where nitrogen is naturally limiting, but carbon is found in abundance. Due to its ability to fix atmospheric nitrogen and produce the bioplastic polyhydroxyalkanoate (PHA), Y88T is well-suited for growth in a nitrogenlimited but carbon-enriched environment. Because of these metabolic abilities, Y88T is of interest as a model organism for PHA production unconstrained by nitrogen-limiting conditions. Growth profiles and PHA production profiles were determined for Y88T under conditions of carbon enrichment, nitrogen sufficiency and depletion to investigate carbon and nitrogen utilisation as well as PHA production in this organism. Also, since the nitrogenase enzyme required for nitrogen fixation is oxygen labile, the effect of DO concentration and the relationship between aerobic metabolism and the nitrogen-fixing and PHA-producing abilities of Y88T was investigated. This study demonstrated: that glucose is the preferred growth substrate for Y88T; that no direct relationship exists between nitrogen fixation and PHB accumulation in Y88T; that Y88T can reliably produce in excess of 80 % of its dry weight as polyhydroxybutyrate (PHB), a type of PHA, from glucose under nitrogenlimiting conditions. Proteomic signatures were determined for the various physiological responses of Y88T to growth, nitrogen utilisation, PHB production and exposure to different levels of DO. More than 250 unique proteins, including the core nitrogen-fixation, PHB-synthetic and glycolytic proteins were identified. Y88T apparently converts glucose to PHB via three interrelated glucose catabolic pathways and proteins likely involved in these pathways were identified. This study revealed that, regardless of growth conditions and despite decreased abundance of the Y88T nitrogenase enzyme, growth and PHB synthesis were not inhibited at DOhigh concentrations. Proteomic characterisation of the Y88T phasin, a PHA granule-associated protein, iii identified an amino-terminal, low complexity alanine and proline rich segment found only in other sphingomonads. The expression level of the Y88T phasin correlated well with PHB yields, suggesting the use of this protein as a biomarker to optimise PHB yield in a production environment. Y88T has the potential to be a useful production strain in pure culture, utilising its natural and robust propensity to metabolise glucose to preferentially produce PHB. Targets for biotechnological improvement and the potential for application of Y88T to biofuel production are discussed.</p>

Novel Approach for the Determination of Nitrogen Fixation in Cyanobacteria

2019 ◽  
Vol 41 (1) ◽  
pp. 105-105
Author(s):  
Sumaira Mazhar Sumaira Mazhar ◽  
Jerry D Cohen and Shahida Hasnain Jerry D Cohen and Shahida Hasnain
Keyword(s):  
Nitrogen Fixation ◽  
Growth Conditions ◽  
Nitrogen Enrichment ◽  
Nitrogen Fixing ◽  
Cultivation Conditions ◽  
Novel Approach ◽  
Detailed Method ◽  
Study Support ◽  
Novel Approaches

Non-heterocystous nitrogen fixing strains of cyanobacteria were screened by their ability to grow in nitrogen deficient media. The selected nitrogen fixing cyanobacterial cells were then cultured in BG11 media supplemented with [15N]-labeled sodium nitrate. Under these growth conditions any organic [14N] found in the cyanobacterial cells would simply come from nitrogen fixation because [15N] was the only available source of nitrogen in the medium. Amino acids extracted after different time periods (after 15, 30, 40, 50 and 60 days of inoculation) were used for the determination of the 14N/15N ratio using GC-MS. Results from the present study support the conclusion that at stationary phase of growth cyanobacterial nitrogen fixation was no longer supplying a significant amount of nitrogen. This approach not only provided a detailed method for the evaluation of the nitrogen fixing potential of the cyanobacteria in culture, but also suggests novel approaches for the assessment of the ability of the strains to provide nitrogen enrichment to plants under co-cultivation conditions.

scholarly journals Essential Genome of the Metabolically Versatile Alphaproteobacterium Rhodopseudomonas palustris

2015 ◽  
Vol 198 (5) ◽  
pp. 867-876 ◽  
Author(s):  
Kieran B. Pechter ◽  
Larry Gallagher ◽  
Harley Pyles ◽  
Colin S. Manoil ◽  
Caroline S. Harwood
Keyword(s):  
Nitrogen Fixation ◽  
Caulobacter Crescentus ◽  
Essential Gene ◽  
Rhodopseudomonas Palustris ◽  
Model Organism ◽  
Growth Conditions ◽  
Essential Genes ◽  
Content Type ◽  
E Coli ◽  
Production Of Hydrogen

ABSTRACTRhodopseudomonas palustrisis an alphaproteobacterium that has served as a model organism for studies of photophosphorylation, regulation of nitrogen fixation, production of hydrogen as a biofuel, and anaerobic degradation of aromatic compounds. This bacterium is able to transition between anaerobic photoautotrophic growth, anaerobic photoheterotrophic growth, and aerobic heterotrophic growth. As a starting point to explore the genetic basis for the metabolic versatility ofR. palustris, we used transposon mutagenesis and Tn-seq to identify 552 genes as essential for viability in cells growing aerobically on semirich medium. Of these, 323 have essential gene homologs in the alphaproteobacteriumCaulobacter crescentus, and 187 have essential gene homologs inEscherichia coli. There were 24R. palustrisgenes that were essential for viability under aerobic growth conditions that have low sequence identity but are likely to be functionally homologous to essentialE. coligenes. As expected, certain functional categories of essential genes were highly conserved among the three organisms, including translation, ribosome structure and biogenesis, secretion, and lipid metabolism.R. palustriscells divide by budding in which a sessile cell gives rise to a motile swarmer cell. Conserved cell cycle genes required for this developmental process were essential in bothC. crescentusandR. palustris. Our results suggest that despite vast differences in lifestyles, members of the alphaproteobacteria have a common set of essential genes that is specific to this group and distinct from that of gammaproteobacteria likeE. coli.IMPORTANCEEssential genes in bacteria and other organisms are those absolutely required for viability.Rhodopseudomonas palustrishas served as a model organism for studies of anaerobic aromatic compound degradation, hydrogen gas production, nitrogen fixation, and photosynthesis. We used the technique of Tn-seq to determine the essential genes ofR. palustrisgrown under heterotrophic aerobic conditions. The transposon library generated in this study will be useful for future studies to identifyR. palustrisgenes essential for viability under specialized growth conditions and also for survival under conditions of stress.

scholarly journals Mechanistic Model for the Coexistence of Nitrogen Fixation and Photosynthesis in Marine Trichodesmium

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Keisuke Inomura ◽  
Samuel T. Wilson ◽  
Curtis Deutsch
Keyword(s):  
Nitrogen Fixation ◽  
Mechanistic Model ◽  
Model Organism ◽  
Extracellular Polysaccharide ◽  
Physiological Model ◽  
Cellular Growth ◽  
Energetic Cost ◽  
Respiratory Protection ◽  
Nitrogen Fixing ◽  
Low Oxygen

ABSTRACT The cyanobacterium Trichodesmium is an important contributor of new nitrogen (N) to the surface ocean, but its strategies for protecting the nitrogenase enzyme from inhibition by oxygen (O2) remain poorly understood. We present a dynamic physiological model to evaluate hypothesized conditions that would allow Trichodesmium to carry out its two conflicting metabolic processes of N2 fixation and photosynthesis. First, the model indicates that managing cellular O2 to permit N2 fixation requires high rates of respiratory O2 consumption. The energetic cost amounts to ∼80% of daily C fixation, comparable to the observed diminution of the growth rate of Trichodesmium relative to other phytoplankton. Second, by forming a trichome of connected cells, Trichodesmium can segregate N2 fixation from photosynthesis. The transfer of stored C to N-fixing cells fuels the respiratory O2 consumption that protects nitrogenase, while the reciprocal transfer of newly fixed N to C-fixing cells supports cellular growth. Third, despite Trichodesmium lacking the structural barrier found in heterocystous species, the model predicts low diffusivity of cell membranes, a function that may be explained by the presence of Gram-negative membrane, production of extracellular polysaccharide substances (EPS), and “buffer cells” that intervene between N2-fixing and photosynthetic cells. Our results suggest that all three factors—respiratory protection, trichome formation, and diffusion barriers—represent essential strategies that, despite their energetic costs, facilitate the growth of Trichodesmium in the oligotrophic aerobic ocean and permit it to be a major source of new reactive nitrogen. IMPORTANCE Trichodesmium is a major nitrogen-fixing cyanobacterium and exerts a significant influence on the oceanic nitrogen cycle. It is also a widely used model organism in laboratory studies. Since the nitrogen-fixing enzyme nitrogenase is extremely sensitive to oxygen, how these surface-dwelling plankton manage the two conflicting processes of nitrogen fixation and photosynthesis has been a long-standing question. In this study, we developed a simple model of metabolic fluxes of Trichodesmium capturing observed daily cycles of photosynthesis, nitrogen fixation, and boundary layer oxygen concentrations. The model suggests that forming a chain of cells for spatially segregating nitrogen fixation and photosynthesis is essential but not sufficient. It also requires a barrier against oxygen diffusion and high rates of oxygen scavenging by respiration. Finally, the model indicates an extremely short life span of oxygen-enabling cells to instantly create a low-oxygen environment upon deactivation of photosynthesis.

Does Dryas integrifolia fix nitrogen?

Botany ◽  
2009 ◽  
Vol 87 (11) ◽  
pp. 1106-1109 ◽  
Author(s):  
John H. Markham
Keyword(s):  
Nitrogen Fixation ◽  
Strong Evidence ◽  
Host Plants ◽  
Symbiotic Nitrogen Fixation ◽  
Growth Conditions ◽  
Nitrogen Fixing ◽  
Purshia Tridentata ◽  
Alnus Viridis ◽  
Vegetatively Propagated Plants ◽  
Different Sources

Symbiotic nitrogen fixation is found in plant taxa that also include non-nitrogen-fixing members. Strong evidence for the occurrence of nitrogen fixation comes from physiological measurements and the identification of the nitrogen fixing symbiont. This evidence has been provided for Dryas drummondii Richardson ex Hook. in the Rosaceae. However, while there have been numerous references to the nitrogen fixing ability of Dryas integrifolia Vahl., they can all be traced to a single report that did not provide strong evidence for nitrogen fixation. My attempts to establish nitrogen fixing nodules on vegetatively propagated plants from the field, or seedlings of D. integrifolia, using three different sources of Frankia , all failed. Since other host plants ( Alnus viridis (Chaix) DC. subsp. crispa (Aiton) Turrill and Purshia tridentata (Pursh.) DC.) did produce nitrogen-fixing nodules under the same growth conditions, the ability of D. integrifolia to fix nitrogen should be considered suspect.

scholarly journals Plasmids Related to the Symbiotic Nitrogen Fixation Are Not Only Cooperated Functionally but Also May Have Evolved over a Time Span in Family Rhizobiaceae

2020 ◽  
Vol 12 (11) ◽  
pp. 2002-2014
Author(s):  
Ling-Ling Yang ◽  
Zhao Jiang ◽  
Yan Li ◽  
En-Tao Wang ◽  
Xiao-Yang Zhi
Keyword(s):  
Nitrogen Fixation ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Symbiotic Nitrogen Fixation ◽  
Gene Families ◽  
Time Span ◽  
Soil Conditions ◽  
Gene Gain ◽  
Nitrogen Fixing ◽  
Pan Genome

Abstract Rhizobia are soil bacteria capable of forming symbiotic nitrogen-fixing nodules associated with leguminous plants. In fast-growing legume-nodulating rhizobia, such as the species in the family Rhizobiaceae, the symbiotic plasmid is the main genetic basis for nitrogen-fixing symbiosis, and is susceptible to horizontal gene transfer. To further understand the symbioses evolution in Rhizobiaceae, we analyzed the pan-genome of this family based on 92 genomes of type/reference strains and reconstructed its phylogeny using a phylogenomics approach. Intriguingly, although the genetic expansion that occurred in chromosomal regions was the main reason for the high proportion of low-frequency flexible gene families in the pan-genome, gene gain events associated with accessory plasmids introduced more genes into the genomes of nitrogen-fixing species. For symbiotic plasmids, although horizontal gene transfer frequently occurred, transfer may be impeded by, such as, the host’s physical isolation and soil conditions, even among phylogenetically close species. During coevolution with leguminous hosts, the plasmid system, including accessory and symbiotic plasmids, may have evolved over a time span, and provided rhizobial species with the ability to adapt to various environmental conditions and helped them achieve nitrogen fixation. These findings provide new insights into the phylogeny of Rhizobiaceae and advance our understanding of the evolution of symbiotic nitrogen fixation.

scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

scholarly journals Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ramona Wördemann ◽  
Lars Wiefel ◽  
Volker F. Wendisch ◽  
Alexander Steinbüchel
Keyword(s):  
Amino Acids ◽  
Dry Weight ◽  
Growth Conditions ◽  
Amino Acid Biosynthesis ◽  
Water Soluble ◽  
Lysine Content ◽  
Polyaspartic Acid ◽  
Acid Biosynthesis ◽  
Cyanophycin Synthetase ◽  
Potential Precursor

AbstractCyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

scholarly journals Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Luz K. Medina-Cordoba ◽  
Aroon T. Chande ◽  
Lavanya Rishishwar ◽  
Leonard W. Mayer ◽  
Lina C. Valderrama-Aguirre ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Nitrogen Fixation ◽  
Plant Growth ◽  
Phosphate Solubilization ◽  
Nitrogen Fixing ◽  
Nitrogen Fixing Bacteria ◽  
Promising Tool ◽  
A Genome ◽  
Plant Growth Promoting ◽  
Growth Promoting

AbstractPrevious studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

Sign in / Sign up

Export Citation Format

Share Document