Acetylene Reduction Assay Research Articles

Deciphering the Nitrogen Fixation Gene Cluster in Vibrio natriegens: A Study on Optimized Expression and Application of Nitrogenase.

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

Azotosporobacter soli gen. nov., sp. nov., a novel nitrogen-fixing bacterium isolated from paddy soil.

A nitrogen-fixing strain designated SG130T was isolated from paddy soil in Fujian Province, China. Strain SG130T was Gram-staining-negative, rod-shaped, and strictly anaerobic. Strain SG130T showed the highest 16S rRNA gene sequence similarities with the type strains Dendrosporobacter quercicolus DSM 1736T (91.7%), Anaeroarcus burkinensis DSM 6283T (91.0%) and Anaerospora hongkongensis HKU 15T (90.9%). Furthermore, the phylogenetic and phylogenomic analysis also suggested strain SG130T clustered with members of the family Sporomusaceae and was distinguished from other genera within this family. Growth of strain SG130T was observed at 25-45°C (optimum 30°C), pH 6.0-9.5 (optimum 7.0) and 0-1% (w/v) NaCl (optimum 0.1%). The quinones were Q-8 and Q-9. The polar lipids were phosphatidylserine (PS), phosphatidylethanolamine (PE), glycolipid (GL), phospholipid (PL) and an unidentified lipid (UL). The major fatty acids (> 10%) were iso-C13:0 3OH (26.6%), iso-C17:1 (15.6%) and iso-C15:1 F (11.4%). The genomic DNA G + C content was 50.7%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T (ANI 68.0% and dDDH 20.3%) were both below the cut-off level for species delineation. The average amino acid identity (AAI) between strain SG130T and the most closely related type strain D. quercicolus DSM 1736T was 63.2%, which was below the cut-off value for bacterial genus delineation (65%). Strain SG130T possessed core genes (nifHDK) involved in nitrogen fixation, and nitrogenase activity (106.38μmol C2H4 g-1 protein h-1) was examined using the acetylene reduction assay. Based on the above results, strain SG130T is confirmed to represent a novel genus of the family Sporomusaceae, for which the name Azotosporobacter soli gen. nov., sp. nov. is proposed. The type strain is SG130T (= GDMCC 1.3312T = JCM 35641T).

DIAZOTROPHIC ENDOPHYTE Klebsiella sp. N5 FROM SORGHUM SHOWS CROSS-COLONIZATION AND PLANT GROWTH PROMOTION IN RICE

Rice necessitates the highest levels of N fertilizers during its cultivation increasing demand for nitrogen fertilizers as well as negatively affecting the environment. Endophytic diazotrophic bacteria were isolated from sorghum, out of 35 isolates Klebsiella sp. N5 showed maximum potential for biological nitrogen fixation. Primary screening of strain N5 was done with acetylene reduction assay indicating dry root activity of 638.55 nmol C2H4 h-1 g-1. That was further confirmed by nifH gene amplification. The strain was identified morphologically, biochemically and 16S rDNA sequencing analysis and identified as Klebsiella sp. strain N5. This strain exhibited other plant growth-promoting (PGP) traits, such as phosphate solubilization, production of indole acetic acid, siderophore activity, ACC deaminase activity, and anti-fungal activity. PGP endophyte N5 was inoculated into rice seedlings to investigate its interaction under axenic conditions and to characterize its ability to colonize non-native host plant rice. Extensive colonization of rice roots was corroborated by Scanning electron microscopy. In-planta experiments with rice seeds inoculated with Klebsiella sp. N5 showed significant improvements in various agronomic traits such as increase in shoot length, root length, fresh and dry weight of root and shoot, chlorophyll content, and nitrogen content of the plant and soil compared to the untreated plants. Klebsiella sp. N5, a cross colonizer from sorghum (C4 plant) to rice (C3 plant) plays a crucial role in facilitating the acquisition of essential resources such as nitrogen, phosphorus, and iron through direct mechanisms. This study expands the horizon for its potential as a valuable bioinoculant for sustainable agricultural practices, particularly in non-native host rice, paving the way for future field trials.

Short-term fate of nitrogen fixed by moss-cyanobacteria associations under different rainfall regimes

Nitrogen (N) fixation by moss-cyanobacteria associations has been recognized as an important N input pathway in many ecosystems from arctic tundra to tropical forests. However, the transfer of fixed N2 from mosses to the soil as well as the effects of rainfall frequency and volume on this N transfer has hardly been studied – even though mosses can leach nutrients upon rewetting. In this study, we investigated the transfer of fixed N2 by moss-cyanobacteria associations in one month under four watering regimes with a combination of high and low volume and frequency. For this, we used two morphologically similar moss species collected from ecosystems with different climate and N availability (subarctic - Hylocomium splendens; and tropical - Thuidium delicatulum). Acetylene reduction assays were conducted as a measure of N2 fixation rates in mosses, and 15N-N2 tracing was used to follow the fixed N2 from moss to the underlying substrate. Nitrogen fixation rates were higher in T. delicatulum than in H. splendens, but rainfall volume and frequency did not show strong effects on N2 fixation rates. Nonetheless, the extent of N leached from mosses was more sensitive to an increase in rainfall volume than to an increase in frequency, and more N was lost from T. delicatulum under high volume precipitation than from H. splendens. Both total nitrogen and 15N enrichment results demonstrate that the fixed N2 was mostly stored in moss tissues with less than 1 % leached to the substrate. Our results show that both moss species retain almost all fixed N2 within their tissues under small rainfall disturbances within one month, while increased N availability under higher precipitation volume renders some moss species an important N source for the soil.

Heterodera glycines HG type 1.2.5.7 causes a decrease in soybean (Glycine max [L.] Merr.) nitrogen fixation and growth variables

ABSTRACT Soybean cyst nematode (SCN), Heterodera glycines, HG type 1.2.5.7 is the most virulent and widespread SCN biotype in the southern states of the USA, but its effect on soybean (Glycine max [L.] Merr.) nitrogen fixation (NF) activity and growth is not well documented. The objectives of this study were to: (1) measure the impact of HG type 1.2.5.7 on NF activity during the early vegetative stage of three soybean genotypes and (2) compare the impact on soybean leaf and root development, and plant growth. The NF activity of two susceptible (S) genotypes (“Ellis” and “Williams 82”) and one resistant (R) genotype (PI 88,788), subjected to a 10,000-egg treatment, was measured using an in-situ flow-through acetylene reduction assay. The average NF activity of the soybean genotypes was decreased by SCN activity. Time points assessment showed that the NF activity was differently impacted among the genotypes. Based on the measured values, the NF activity was decreased by 30% in SCN infected plants of Williams 82 but that of PI 88,788 and Ellis was affected. Likewise, soybean leaf color and leaf area were affected by SCN infection, but there was no impact on root length and root area. These results showed that SCN HG type 1.2.5.7 can exhibit varying negative impact on NF and shoot growth of S soybean genotypes, while R genotypes may be unaffected. Thus, indicating genetic resistance continues to be a critical tool for the management of the SCN.

Nitrogen Fixing Activity of Endophytic Bacteria Associated with Kalanchoe pinnata (Lam.) and its effect on Zea mays

ABSTRACT: Nitrogen is essentially required for the plant growth as well as productivity. Plants take nitrogen in the form of ammonia or nitrate either from soil or fertilizer. There are nitrogen fixing bacteria plays a vital role to supply atmospheric nitrogen to plants where plants do not obtain from soil. Apart from soil bacteria, endophytic bacteria which living inside the plant tissues can improve crop productivity and soil health sustainably through biological nitrogen fixation and act as a potential replacement for chemical fertilizers in agriculture. This study was conducted to determine the nitrogen fixing activity of isolated endophytic bacteria from Kalanchoe pinnata (Lam.). The isolated endophytes were subjected to molecular confirmation and evaluated for ammonia production, Acetylene Reduction Assay (ARA), nif gene amplification and analysis of growth parameters in Zea mays using pot culture assay. The data were analyzed using SPSS ver.16. In this study, Bacillus thuringiensis, Bacillus paranthracis, Staphylococcus xylosus and Bacillus cereus were isolated from the leaves of Kalachoe pinnata (Lam). They were confirmed using 16SrRNA sequencing. All the endophytic bacteria were positive for ammonia production and ARA. The percentage of nitrogen produced was 32.8 % (B. thuringiensis), 65.7% (B. paranthracis), 80.7% (S. xylosus) and 45.2% (B. cereus).The presence of nif gene was confirmed through the PCR amplification of a 550-580bp fragment of the gene. Pot culture assay of Zea mays were observed with significant improvement in S. xylosus followed by B. paranthracis inoculated pots. The presence of the nitrogenase enzyme and the nif gene in these endophytic bacteria allows them to fix atmospheric nitrogen to meet plant nitrogen demands, resulting in a symbiotic relationship with agricultural crops.

The Feather Moss Hylocomium splendens Affects the Transcriptional Profile of a Symbiotic Cyanobacterium in Relation to Acquisition and Turnover of Key Nutrients

Moss-cyanobacteria symbioses were proposed to be based on nutrient exchange, with hosts providing C and S while bacteria provide N, but we still lack understanding of the underlying molecular mechanisms of their interactions. We investigated how contact between the ubiquitous moss Hylocomium splendens and its cyanobiont affects nutrient-related gene expression of both partners. We isolated a cyanobacterium from H. splendens and co-incubated it with washed H. splendens shoots. Cyanobacterium and moss were also incubated separately. After 1 week, we performed acetylene reduction assays to estimate N2 fixation and RNAseq to evaluate metatranscriptomes. Genes related to N2 fixation and the biosynthesis of several amino acids were up-regulated in the cyanobiont when hosted by the moss. However, S-uptake and the biosynthesis of the S-containing amino acids methionine and cysteine were down-regulated in the cyanobiont while the degradation of selenocysteine was up-regulated. In contrast, the number of differentially expressed genes in the moss was much lower, and almost no transcripts related to nutrient metabolism were affected. It is possible that, at least during the early stage of this symbiosis, the cyanobiont receives few if any nutrients from the host in return for N, suggesting that moss–cyanobacteria symbioses encompass relationships that are more plastic than a constant mutualist flow of nutrients.

Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL− 1·h− 1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Diazotrophic nitrogen fixation through aerial roots occurs in Avicennia marina: implications for adaptation of mangrove plant growth to low-nitrogen tidal flats.

Nitrogen limitation of primary production is common in coastal ecosystems. Mangrove trees maintain high levels of nitrogen fixation around their roots. The interior aerial space of mangrove roots, in which atmospheric gas is supplied through lenticels, could be efficient sites for nitrogen fixation. We measured tidal variations of partial pressure of N2 in root aerenchyma and conducted field experiments using 15 N2 as a tracer to track N2 movement through aerial roots of Avicennia marina. We used the acetylene reduction assay to identify the root parts harboring diazotrophs. The nitrogenase activity and estimated nitrogen fixation through aerenchyma were higher in pneumatophores and absorbing roots than in cable roots. Positive correlations between root nitrogen contents and turnover rates of root nitrogen derived from N2 through aerenchyma suggested that the internal supply of N2 to diazotrophs could be the main source for nitrogen assimilation by A. marina roots. Our results confirmed that N2 is supplied to diazotrophs through aerial roots and that nitrogen fixation occurs in A. marina roots. The aerial root structures, which occur across families of mangrove plants, could be an adaptation to survival in not only low-oxygen environments but also tidal flats with little plant-available nitrogen.

Increasing plant species diversity suppresses free‐living N2 fixation in litter and soil of a subtropical karst forest

Abstract Free‐living N2 fixation (FNF) is an important source of bioavailable nitrogen for forests. Though increasing plant species diversity (PSD) benefits soil nitrogen accumulation, how it impacts FNF rate has not been explored. Forty‐five plots covering a PSD gradient were selected in a subtropical karst forest, southwest China. FNF rates in leaf litter and soil were measured using acetylene reduction assay calibrated with a 15N2 fixation method. Diazotrophic community, litter and soil properties were determined as well. The nifH gene abundance was not significantly altered in litter but was significantly decreased in soil by higher PSD. Diazotrophic community's Shannon diversity was significantly increased by higher PSD in both litter and soil. Increasing PSD inhibited litter FNF rate via aggravating phosphorus limitation and decreasing the relative abundance of Betaproteobacteria and Deltaproteobacteria. For the mineral soil horizon, increasing PSD suppressed FNF rate via decreasing nifH gene abundance caused by elevated N availability and lowered phosphorus and iron availability or through decreasing the relative abundance of Deltaproteobacteria but increasing that of Betaproteobacteria. Our findings, for the first time, reveal the microbial and abiotic controls on FNF in litter and soil in response to PSD, and hence benefit the prediction of nitrogen input via biological N2 fixation under changes in PSD. Read the free Plain Language Summary for this article on the Journal blog.

Nitrogen fixation and transcriptome of a new diazotrophic Geomonas from paddy soils.

The ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.

Nitrogen fixation associated with epiphytes on the seagrass Zostera marina in a temperate lagoon with moderate to high nitrogen loads

AbstractAs part of a long-term study on the effects of nitrogen (N) loading in a shallow temperate lagoon, we measured rates of N2 fixation associated with seagrass (Zostera marina) epiphytes during the summer from 2005 to 2019, at two sites along a gradient from where high N groundwater enters the system (denoted SH) to a more well-flushed outer harbor (OH). The data presented here are the first such long-term N2 fixation estimates for any seagrass system and one of the very few reported for the phyllosphere in a temperate system. Mean daily N2 fixation was estimated from light and dark measurements using the acetylene reduction assay intercalibrated using both incorporation of 15N2 into biomass and a novel application of the N2:Ar method. Surprisingly, despite a large inorganic N input from a N-contaminated groundwater plume, epiphytic N2 fixation rates were moderately to very high for a seagrass system (OH site 14-year mean of 0.94 mmol N m−2 d−1), with the highest rates (2.6 mmol N m−2 d−1) measured at the more N-loaded eutrophic site (SH) where dissolved inorganic N was higher and soluble reactive phosphorus was lower than in the better-flushed OH. Over 95% of the total N2 fixation measured was in the light, suggesting the importance of cyanobacteria in the epiphyte assemblages. We observed large inter-annual variation both within and across the two study sites (range from 0.1 to 2.6 mmol N fixed m−2 d−1), which we suggest is in part related to climatic variation. We estimate that input from phyllosphere N2 fixation over the study period contributes on average an additional 20% to the total daily N load per area within the seagrass meadow.

(Invited) Using the Model Bacterium Geobacter Sulfurreducens to Understand Microbial Electrochemical Conversion of Nitrogen Gas into Ammonium

There is a growing need to decrease reliance on the Haber-Bosch process. To convert nitrogen gas (N2) into ammonia (NH3) for fertilizers, this temperature- and pressure-intensive process consumes roughly 2–3% of global energy and releases over 740 million tons of CO2 each year. Many microorganisms naturally convert or “fix” N2 into ammonium (NH4 +) at room temperature and pressure using an enzyme called the nitrogenase. Attempts to increase NH4 + yields from microorganisms through genetic engineering are seeing signs of success, but we lack methods to increase NH4 + generation rates and overcome one of the largest challenges: O2 gas. Increasing O2 gas concentrations typically increases metabolic rates of aerobic microorganisms; however, in the case of microorganisms that fix N2 (called diazotrophs), O2 can shut down the nitrogenase and stop cell growth. To overcome these challenges, we are using electricity to drive N2 fixation in exoelectrogenic bacteria. These bacteria have the unique ability to naturally transfer electrons to anode electrodes in microbial electrochemical technologies (METs). Increasing the voltage applied to METs increases the metabolic rates of exoelectrogens. The anode chamber is kept anaerobic, which eliminates O2-driven inhibition of the nitrogenase. We previously showed that the N2 fixation rates of a mixed microbial community in a MET increased more than three times when the applied whole-cell potential increased from 0.7 V to 1.0 V. By adding a chemical inhibitor that prevented the incorporation of NH4 + into larger biomolecules, NH4 + was excreted by the cells and into the medium. Based on the acetylene reduction assay (a proxy for N2 fixation), we estimated that we recovered about 10% of the theoretical NH4 +generated by the cells. If close to 100% of the theoretical NH4 + could be excreted from the cells and recovered, we predicted an energy demand of around 3 MJ/mol-NH4 +, which is close to the range of value reported for the Haber-Bosch process. To develop METs that generate NH4 + at competitive production rates and energy demands, we are now focusing on the model exoelectrogenic diazotroph Geobacter sulfurreducens. Using a single organism with a fully sequenced genome allows us to understand how the N2 fixation process responds to electrochemical variables. Towards these efforts, we conducted a transcriptomic analysis of G. sulfurreducens at two fixed anode potentials: +0.15 V and −0.15 V. This approach revealed which genes are turned on and off in response to these potentials. The presence of NH4 + decreased the expression of genes associated with N2 fixation which was expected due to the known sensitivity of nitrogenases to NH4 +. On the other hand, the two anode potentials had a dramatic and unexpected impact on the expression levels of N2 fixation genes. At −0.15 V, nitrogenase genes were significantly up-regulated, as were genes associated with NH4 + uptake and transport, such as glutamine and glutamate synthetases. Considering that −0.15 V provides less energy to the cells relative to +0.15 V (based on thermodynamic predictions), the results suggest that the cells responded to this highly energy-constrained environment by increasing expression of N2 fixation pathways. Based on our new knowledge of how G. sulfurredcuens regulates N2 fixation and NH4 + production, we have developed new strains of G. sulfurreducens that can excrete NH4 + during N2 fixation. The new engineering toolkit that we have created in this bacterium can now be leveraged to maximize NH4 + production in METs.

Nitrogen fixation in the stag beetle, Ceruchus piceus (Coleoptera: Lucanidae): could insects contribute more to ecosystem nitrogen budgets than previously thought?

Nitrogen (N) is a key nutrient required by all living organisms for growth and development, but is a limiting resource for many organisms. Organisms that feed on material with low N content, such as wood, might be particularly prone to N limitation. In this study, we investigated the degree to which the xylophagous larvae of the stag beetle Ceruchus piceus (Weber) use associations with N-fixing bacteria to acquire N. We paired acetylene reduction assays by cavity ring-down absorption spectroscopy (ARACAS) with 15N2 incubations to characterize rates of N fixation within C. piceus. Not only did we detect significant N fixation activity within C. piceus larvae, but we calculated a rate that was substantially higher than most previous reports for N fixation in insects. While taking these measurements, we discovered that N fixation within C. piceus can decline rapidly in a lab setting. Consequently, our results demonstrate that previous studies, which commonly keep insects in the lab for long periods of time prior to and during measurement, may have systematically under-reported rates of N fixation in insects. This suggests that within-insect N fixation may contribute more to insect nutrition and ecosystem-scale N budgets than previously thought.

Carnivorous Nepenthes Pitchers with Less Acidic Fluid House Nitrogen-Fixing Bacteria.

Carnivorous pitcher plants are uniquely adapted to nitrogen limitation, using pitfall traps to acquire nutrients from insect prey. Pitcher plants in the genus Sarracenia may also use nitrogen fixed by bacteria inhabiting the aquatic microcosms of their pitchers. Here, we investigated whether species of a convergently evolved pitcher plant genus, Nepenthes, might also use bacterial nitrogen fixation as an alternative strategy for nitrogen capture. First, we constructed predicted metagenomes of pitcher organisms from three species of Singaporean Nepenthes using 16S rRNA sequence data and correlated predicted nifH abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive nifH PCR tests. Third, we analyzed nifH in eight shotgun metagenomes from four additional Bornean Nepenthes species. Finally, we conducted an acetylene reduction assay using greenhouse-grown Nepenthes pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in Nepenthes pitcher fluid. Variation in nifH from wild samples correlates with Nepenthes host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous Nepenthes digestive enzymes are most active at low fluid pH. We hypothesize Nepenthes species experience a trade-off in nitrogen acquisition; when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants take up more nitrogen via bacterial nitrogen fixation. IMPORTANCE Plants use different strategies to obtain the nutrients that they need to grow. Some plants access their nitrogen directly from the soil, while others rely on microbes to access the nitrogen for them. Carnivorous pitcher plants generally trap and digest insect prey, using plant-derived enzymes to break down insect proteins and generate a large portion of the nitrogen that they subsequently absorb. In this study, we present results suggesting that bacteria living in the fluids formed by Nepenthes pitcher plants can fix nitrogen directly from the atmosphere, providing an alternative pathway for plants to access nitrogen. These nitrogen-fixing bacteria are only likely to be present when pitcher plant fluids are not strongly acidic. Interestingly, the plant's enzymes are known to be more active under strongly acidic conditions. We propose a potential trade-off where pitcher plants sometimes access nitrogen using their own enzymes to digest prey and at other times take advantage of bacterial nitrogen fixation.

A novel strictly anaerobic sulfate-reducing diazotrophic bacterium Fundidesulfovibrio terrae sp. nov., isolated from paddy soil.

A strictly anaerobic sulfate-reducing strain, designated SG127T, was isolated from paddy soil. SG127T showed the highest 16S rRNA gene sequence similarity to the type strain of Fundidesulfovibrio magnetotacticus (98.2 %). A phylogenetic tree based on 16S rRNA gene sequences indicated that SG127T clustered with members of the genus Fundidesulfovibrio. Growth of SG127T was observed at 20-37 °C (optimum, 30 °C), pH 5.5-9.0 (optimum, 7.0-8.0) and with 0-0.2 % (w/v) NaCl (optimally without NaCl). SG127T contained MK-7 as the only menaquinone and anteiso-C15 : 0, anteiso-C17 : 1ω9c, C18 : 0, iso-C14 : 0, iso-C15 : 0, iso-C16:0, iso-C16 : 1H, iso-C18 : 1H and summed feature nine as the major fatty acids. The genomic DNA G+C content of SG127T was 64.6 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SG127T and the closely related Fundidesulfovibrio magnetotacticus were 78.5% and 23.2 %, respectively, which were lower than the cut-off values (ANI 95-96% and dDDH 70 %) for prokaryotic species delineation. SG127T had desulfoviridin, possessed nitrogen fixation genes (nifHDK) and actively fixed nitrogen according to the acetylene reduction assay. On the basis of these results, strain SG127T represents a novel species of the genus Fundidesulfovibrio, for which the name Fundidesulfovibrio terrae sp. nov. is proposed. The type strain is SG127T (= GDMCC 1.3137T = JCM 35589T).

Sulfurospirillum oryzae sp. nov., A Novel Nitrogen-Fixing Bacterium Isolated from Paddy Soil

An anaerobic, Gram-staining-negative, rod shaped, nitrogen-fixing strain designed SG202T, was isolated from paddy soil collected from Fujian Province in China. Strain SG202T showed the highest 16S rRNA gene sequence similarity with the type strain Sulfurospirillum multivorans DSM 12446T (98.5%). Phylogenetic trees based on 16S rRNA gene sequences and conserved core genes from genomes indicated that strain SG202T branched with members of the genus Sulfurospirillum. Growth was observed at 25-37 °C (optimum 30 °C), pH 6.0-10.5 (optimum 7.5), and 0-0.6% (w/v) NaCl (optimum 0.2%). Strain SG202T contained MK-6 as the menaquinone and C16:1ω7c (40.6%), C16:0 (33.3%), C18:1ω7c (13.6%) and C14:0 (9.0%) as the major fatty acids. The genomic DNA G+C content was 39.0%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG202T and its closely related species S. multivorans DSM 12446T, Sulfurospirillum halorespirans DSM 13726T, Sulfurospirillum arsenophilum DSM 10659T and Sulfurospirillum diekertiae ACSDCET were 81.3, 81.5, 84.4, 82.2% and 24.5, 24.5, 27.9, 25.2%, respectively. All these values were lower than the recommended species delineation thresholds of ANI (95-96%) and dDDH (70%). Strain SG202T possessed core genes (nifHDK) of nitrogen fixation, and nitrogenase activities (3470.45 μmol C2H4 g-1 protein h-1) was examined using the acetylene reduction assay. Based on the observed physiological properties, chemotaxonomic characteristics and genome analysis, strain SG202T is recognized as a novel species of the genus Sulfurospirillum, for which the name Sulfurospirillum oryzae sp. nov. is proposed. The type strain is SG202T (= GDMCC 1.3379T= JCM 35596T).

Changes in the microbiome of the sea anemone Exaiptasia diaphana during bleaching from short-term thermal elevation

We examined the response of microbial communities in the model sea anemone Exaiptasia diaphana (Aiptasia) to short-term thermal elevation. Through 16S rRNA gene sequencing, we characterized the microbiomes of symbiotic (with algal symbionts) and aposymbiotic (bleached) anemones under ambient (27°C) and heat-stressed (34°C) conditions for 8-10 days, using both replicated endpoint and non-replicated time-course approaches. Consistent with prior studies, we observed a stable abundance of bacteria from the families Alteromonadaceae and Rhodobacteraceae, though with wide variation among individual anemones. We observed that symbiotic state conferred a larger impact on the microbiome than heat stress, implying the microbiome may play a metabolic role in the maintenance of cnidarian-dinoflagellate symbiosis. In particular, Pelobacter, an anaerobic sulfate reducer that is also a potential nitrogen fixer, was present only in symbiotic anemones, and its abundance decreased with initial exposure to 34°C, but recovered after 7 days. In aposymbiotic anemones, the added heat stress appeared to result in a large increase of rare bacterial taxa, which included potential pathogens such as Vibrio following bleaching. We also observed several archaea, the first reported for this model, but only in the seawater surrounding aposymbiotic Aiptasia, where abundance increased dramatically following heat stress. We further explored the diazotrophic (nitrogen fixation) potential of diverse bacteria associated with symbiotic and aposymbiotic Aiptasia, under both ambient and heat-stressed conditions, using nifH-PCR and qPCR and the acetylene reduction assay (ARA). In contrast to some stony corals, nifH was barely expressed in both anemone types, and under ambient conditions, diazotrophic activity was not detectable via ARA. Thus, although this research contributes to the growing knowledge of the bacterial community associated with a prominent model used in coral-symbiosis research, our results also suggest using caution when making direct comparisons between Aiptasia and different coral species in microbiome studies.

Acetylene Reduction Assay: A Measure of Nitrogenase Activity in Plants and Bacteria.

Nitrogen is one of the most abundant elements in the biosphere, but its gaseous form is not biologically available to many organisms, including plants and animals. Diazotrophic microorganisms can convert atmospheric nitrogen into ammonia, a form that can be absorbed by plants in a process called biological nitrogen fixation (BNF). BNF is catalyzed by the enzyme nitrogenase, which not only reduces N2 to NH3 , but also reduces other substrates such as acetylene. The acetylene reduction assay (ARA) can be used to measure nitrogenase activity in diazotrophic organisms, either in symbiotic associations or in their free-living state. The technique uses gas chromatography to measure the reduction of acetylene to ethylene by nitrogenase in a simple, quick, and inexpensive manner. Here, we demonstrate how to: prepare nodulated soybean plants and culture free-living Azospirillum brasilense for the ARA, use the gas chromatograph to detect the ethylene formed, and calculate the nitrogenase activity based on the peaks generated by the chromatograph. The methods shown here using example organisms can be easily adapted to other nodulating plants and diazotrophic bacteria. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acetylene reduction assay in root nodules Basic Protocol 2: Acetylene reduction assay using diazotrophic bacteria Basic Protocol 3: Calculation of nitrogenase activity Support Protocol 1: Production of acetylene from calcium carbide Support Protocol 2: Calibration of the gas chromatograph Support Protocol 3: Total protein quantification.

A Novel Nitrogen-Fixing Bacterium Raoultella electrica Isolated from the Midgut of the Leafhopper Recilia dorsalis.

Nitrogen is a crucial element for the growth and development of insects, but herbivorous insects often suffer from nitrogen nutrition deficiencies in their diets. Some symbiotic microorganisms can provide insect hosts with nitrogen nutrition through nitrogen fixation. Extensive research has clearly demonstrated the process of nitrogen fixation by symbiotic microorganisms in termites, while evidence supporting the occurrence and significance of nitrogen fixation in the diets of the Hemiptera is less conclusive. In this study, we isolated a strain of R. electrica from the digestive tract of a leafhopper, R. dorsalis, and found that it had nitrogen-fixing capabilities. Fluorescence in situ hybridization results showed that it was located in the gut of the leafhopper. Genome sequencing revealed that R. electrica possessed all the genes required for nitrogen fixation. We further evaluated the growth rate of R. electrica in nitrogen-containing and nitrogen-free media and measured its nitrogenase activity through an acetylene reduction assay. The findings of these studies could shed light on how gut microbes contribute to our understanding of nitrogen fixation.