Acetylene Tunes Microbial Growth During Aerobic Cometabolism of Trichloroethene.

Microbial aerobic cometabolism is a possible treatment approach for large, dilute trichloroethene (TCE) plumes at groundwater contaminated sites. Rapid microbial growth and bioclogging pose a persistent problem in bioremediation schemes. Bioclogging reduces soil porosity and permeability, which negatively affects substrate distribution and contaminant treatment efficacy while also increasing the operation and maintenance costs of bioremediation. In this study, we evaluated the ability of acetylene, an oxygenase enzyme-specific inhibitor, to decrease biomass production while maintaining aerobic TCE cometabolism capacity upon removal of acetylene. We first exposed propane-metabolizing cultures (pure and mixed) to 5% acetylene (v v-1) for 1, 2, 4, and 8 d and we then verified TCE aerobic cometabolic activity. Exposure to acetylene overall decreased biomass production and TCE degradation rates while retaining the TCE degradation capacity. In the mixed culture, exposure to acetylene for 1-8 d showed minimal effects on the composition and relative abundance of TCE cometabolizing bacterial taxa. TCE aerobic cometabolism and incubation conditions exerted more notable effects on microbial ecology than did acetylene. Acetylene appears to be a viable approach to control biomass production that may lessen the likelihood of bioclogging during TCE cometabolism. The findings from this study may lead to advancements in aerobic cometabolism remediation technologies for dilute plumes.

Acetylenotrophic and Diazotrophic Bradyrhizobium sp. Strain I71 from TCE-Contaminated Soils.

Acetylene (C2H2) is a molecule rarely found in nature, with very few known natural sources, but acetylenotrophic microorganisms can use acetylene as their primary carbon and energy source. As of 2018 there were 15 known strains of aerobic and anaerobic acetylenotrophs; however, we hypothesize there may yet be unrecognized diversity of acetylenotrophs in nature. This study expands the known diversity of acetylenotrophs by isolating the aerobic acetylenotroph, Bradyrhizobium sp. strain I71, from trichloroethylene (TCE)-contaminated soils. Strain I71 is a member of the class Alphaproteobacteria and exhibits acetylenotrophic and diazotrophic activities, the only two enzymatic reactions known to transform acetylene. This unique capability in the isolated strain may increase the genus' economic impact beyond agriculture as acetylenotrophy is closely linked to bioremediation of chlorinated contaminants. Computational analyses indicate that the Bradyrhizobium sp. strain I71 genome contains 522 unique genes compared to close relatives. Moreover, applying a novel hidden Markov model of known acetylene hydratase (AH) enzymes identified a putative AH enzyme. Protein annotation with I-TASSER software predicted the AH from the microbe Syntrophotalea acetylenica as the closest structural and functional analog. Furthermore, the putative AH was flanked by horizontal gene transfer (HGT) elements, like that of AH in anaerobic acetylenotrophs, suggesting an unknown source of acetylene or acetylenic substrate in the environment that is selecting for the presence of AH. IMPORTANCE The isolation of Bradyrhizobium strain I71 expands the distribution of acetylene-consuming microbes to include a group of economically important microorganisms. Members of Bradyrhizobium are well studied for their abilities to improve plant health and increase crop yields by providing bioavailable nitrogen. Additionally, acetylene-consuming microbes have been shown to work in tandem with other microbes to degrade soil contaminants. Based on genome, cultivation, and protein prediction analysis, the ability to consume acetylene is likely not widespread within the genus Bradyrhizobium. These findings suggest that the suite of phenotypic capabilities of strain I71 may be unique and make it a good candidate for further study in several research avenues.

ATP-dependent substrate reduction at an [Fe8S9] double-cubane cluster.

Chemically demanding reductive conversions in biology, such as the reduction of dinitrogen to ammonia or the Birch-type reduction of aromatic compounds, depend on Fe/S-cluster-containing ATPases. These reductions are typically catalyzed by two-component systems, in which an Fe/S-cluster-containing ATPase energizes an electron to reduce a metal site on the acceptor protein that drives the reductive reaction. Here, we show a two-component system featuring a double-cubane [Fe8S9]-cluster [{Fe4S4(SCys)3}2(µ2-S)]. The double-cubane-cluster-containing enzyme is capable of reducing small molecules, such as acetylene (C2H2), azide (N3-), and hydrazine (N2H4). We thus present a class of metalloenzymes akin in fold, metal clusters, and reactivity to nitrogenases.

Double-Cubane [8Fe9S] Clusters: A Novel Nitrogenase-Related Cofactor in Biology.

Three different types of electron-transferring metallo-ATPases are able to couple ATP hydrolysis to the reduction of low-potential metal sites, thereby energizing an electron. Besides the Fe-protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co-corrinoid-containing proteins (RACE-type activators) or a second iron-sulfur cluster-containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double-cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP-dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double-cubane cofactor are analogous in function and share some structural features with nitrogenases.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Glutathione is Involved in Detoxification of Peroxide and Root Nodule Symbiosis of Mesorhizobium huakuii.

Legumes interact with symbiotic rhizobia to produce nitrogen-fixation root nodules under nitrogen-limiting conditions. The contribution of glutathione (GSH) to this symbiosis and anti-oxidative damage was investigated using the M. huakuii gshB (encoding GSH synthetase) mutant. The gshB mutant grew poorly with different monosaccharides, including glucose, sucrose, fructose, maltose, or mannitol, as sole sources of carbon. The antioxidative capacity of gshB mutant was significantly decreased by these treatments with H2O2 under the lower concentrations and cumene hydroperoxide (CUOOH) under the higher concentrations, indicating that GSH plays different roles in response to organic peroxide and inorganic peroxide. The gshB mutant strain displayed no difference in catalase activity, but significantly lower levels of the peroxidase activity and the glutathione reductase activity than the wild type. The same level of catalase activity could be associated with upregulation of the transcriptional activity of the catalase genes under H2O2-induced conditions. The nodules infected by the gshB mutant were severely impaired in abnormal nodules, and showed a nodulation phenotype coupled to a 60% reduction in the nitrogen fixation capacity. A 20-fold decrease in the expression of two nitrogenase genes, nifH and nifD, is observed in the nodules induced by gshB mutant strain. The symbiotic deficiencies were linked to bacteroid early senescence.

Structural Mimics of Acetylene Hydratase: Tungsten Complexes Capable of Intramolecular Nucleophilic Attack on Acetylene.

Bioinspired complexes employing the ligands 6-tert-butylpyridazine-3-thione (SPn) and pyridine-2-thione (SPy) were synthesized and fully characterized to mimic the tungstoenzyme acetylene hydratase (AH). The complexes [W(CO)(C2 H2 )(CHCH-SPy)(SPy)] (4) and [W(CO)(C2 H2 )(CHCH-SPn)(SPn)] (5) were formed by intramolecular nucleophilic attack of the nitrogen donors of the ligand on the coordinated C2 H2 molecule. Labelling experiments using C2 D2 with the SPy system revealed the insertion reaction proceeding via a bis-acetylene intermediate. The starting complex [W(CO)(C2 H2 )(SPy)2 ] (6) for these studies was accessed by the new acetylene precursor mixture [W(CO)(C2 H2 )n (MeCN)3-n Br2 ] (n=1 and 2; 7). All complexes represent rare examples in the field of W-C2 H2 chemistry with 4 and 5 being the first of their kind. In the ongoing debate on the enzymatic mechanism, the findings support activation of acetylene by the tungsten center.

Evaluating the metabolic perturbations in Mangifera indica (mango) ripened with various ripening agents/practices through gas chromatography - mass spectrometry based metabolomics.

Mangifera indica L. (mango) is said to be the king of fruits due to its rich nutritional properties and mainly originates from the Indian sub-continent. The consumption pattern of the mangoes has increased drastically, due to which, many ripening practices/agents were used to make it ready-to-eat fruit or juice for the consumers. The fruit quality and metabolic composition are said to be altered due to different ripening agents/practices. The present communication mainly deals to understand the metabolic perturbations in mango fruits due to different ripening practices/agents (room temperature ripening, ethylene, and calcium carbide) using gas chromatography - mass spectrometry based metabolomics. The partial least square-discriminant analysis has found 16 differential metabolites for different ripening agents/practices which are belong to the classes of amino acids, fatty acids, sugars, and polyols. Four metabolic pathways were found to alter in the fruit metabolome due to different ripening agents/practices. Fructose, glucose, and galactose were found to be significantly up-regulated due to calcium carbide ripening in comparison to other ripening agents/practices. Overall findings from the present study advocates that mass spectrometry based metabolomics can be valuable tool to understand the fruit quality and safety with respect to consumer health.

Cyclotrimerization of phenylacetylene catalyzed by a cobalt half-sandwich complex embedded in an engineered variant of transmembrane protein FhuA.

An (η5-cyclopentadienyl)cobalt(i) complex was covalently incorporated into an engineered variant of the transmembrane protein ferric hydroxamate uptake protein component: A, FhuA ΔCVFtev, using a thiol-ene reaction. A CD spectrum shows the structural integrity of the biohybrid catalyst. MALDI-TOF of the segment containing the anchoring site for the cobalt complex Cys545 confirmed successful conjugation. This biohybrid catalyst catalyzed the cyclotrimerization of phenylacetylene to give a mixture of regioisomeric 1,2,4- and 1,3,5-triphenylbenzene in aqueous medium.

Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93.

Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days.IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.

Molybdenum and phosphorus limitation of moss-associated nitrogen fixation in boreal ecosystems.

Biological nitrogen fixation (BNF) performed by moss-associated cyanobacteria is one of the main sources of new nitrogen (N) input in pristine, high-latitude ecosystems. Yet, the nutrients that limit BNF remain elusive. Here, we tested whether this important ecosystem function is limited by the availability of molybdenum (Mo), phosphorus (P), or both. BNF in dominant mosses was measured with the acetylene reduction assay (ARA) at different time intervals following Mo and P additions, in both laboratory microcosms with mosses from a boreal spruce forest and field plots in subarctic tundra. We further used a 15 N2 tracer technique to assess the ARA to N2 fixation conversion ratios at our subarctic site. BNF was up to four-fold higher shortly after the addition of Mo, in both the laboratory and field experiments. A similar positive response to Mo was found in moss colonizing cyanobacterial biomass. As the growing season progressed, nitrogenase activity became progressively more P limited. The ARA : 15 N2 ratios increased with increasing Mo additions. These findings show that N2 fixation activity as well as cyanobacterial biomass in dominant feather mosses from boreal forests and subarctic tundra are limited by Mo availability.

Acetylene Fuels TCE Reductive Dechlorination by Defined Dehalococcoides/Pelobacter Consortia.

Acetylene (C2H2) can be generated in contaminated groundwater sites as a consequence of chemical degradation of trichloroethene (TCE) by in situ minerals, and C2H2 is known to inhibit bacterial dechlorination. In this study, we show that while high C2H2 (1.3 mM) concentrations reversibly inhibit reductive dechlorination of TCE by Dehalococcoides mccartyi isolates as well as enrichment cultures containing D. mccartyi sp., low C2H2 (0.4 mM) concentrations do not inhibit growth or metabolism of D. mccartyi. Cocultures of Pelobacter SFB93, a C2H2-fermenting bacterium, with D. mccartyi strain 195 or with D. mccartyi strain BAV1 were actively sustained by providing acetylene as the electron donor and carbon source while TCE or cis-DCE served as the electron acceptor. Inhibition by acetylene of reductive dechlorination and methanogenesis in the enrichment culture ANAS was observed, and the inhibition was removed by adding Pelobacter SFB93 into the consortium. Transcriptomic analysis of D. mccartyi strain 195 showed genes encoding for reductive dehalogenases (e.g., tceA) were not affected during the C2H2-inhibition, while genes encoding for ATP synthase, biosynthesis, and Hym hydrogenase were down-regulated during C2H2 inhibition, consistent with the physiological observation of lower cell yields and reduced dechlorination rates in strain 195. These results will help facilitate the optimization of TCE-bioremediation at contaminated sites containing both TCE and C2H2.

Increased heterocyst frequency by patN disruption in Anabaena leads to enhanced photobiological hydrogen production at high light intensity and high cell density.

The effects of increasing the heterocyst-to-vegetative cell ratio on the nitrogenase-based photobiological hydrogen production by the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were studied. Using the uptake hydrogenase-disrupted mutant (ΔHup) as the parent, a deletion-insertion mutant (PN1) was created in patN, known to be involved in heterocyst pattern formation and leading to multiple singular heterocysts (MSH) in Nostoc punctiforme strain ATCC 29133. The PN1 strain showed heterocyst differentiation but failed to grow in medium free of combined-nitrogen; however, a spontaneous mutant (PN22) was obtained on prolonged incubation of PN1 liquid cultures and was able to grow robustly on N2. The disruption of patN was confirmed in both PN1 and PN22 by PCR and whole genome resequencing. Under combined-nitrogen limitation, the percentage of heterocysts to total cells in the PN22 filaments was 13-15 and 16-18% under air and 1% CO2-enriched air, respectively, in contrast to the parent ΔHup which formed 6.5-11 and 9.7-13% heterocysts in these conditions. The PN22 strain exhibited a MSH phenotype, normal diazotrophic growth, and higher H2 productivity at high cell concentrations, and was less susceptible to photoinhibition by strong light than the parent ΔHup strain, resulting in greater light energy utilization efficiency in H2 production on a per unit area basis under high light conditions. The increase in MSH frequency shown here appears to be a viable strategy for enhancing H2 productivity by outdoor cultures of cyanobacteria in high-light environments.

Nitrogen fixing potential of various heterotrophic Bacillus strains from a tropical estuary and adjacent coastal regions.

In the present study, we report the nitrogen fixing potential of heterotrophic diazotrophs isolated from a tropical estuary and adjacent coastal sea. Results of the study revealed that most of the species that are capable of fixing nitrogen in the study area belongs to the genus Bacillus. The isolates from the estuary showed maximum homology with Bacillus megaterium, B. cereus, B. safencis, B. licheniformis, B. aerophilus, B. oceanisediminis, B. flexus, B. aquimaris, B. vietnamensis, and B. subterraneaus, whereas the diazotrophic isolates from coastal samples were closely related to B. subtilis, B. megaterium, B. circulans, B. aerophilus, B. flexus, and B. oceanisediminis. Experimental studies to determine the nitrogen fixation potential of isolates revealed considerable variation among different strains and the highest nitrogen fixing potential was recorded in B. megaterium (210.05 ± 7.0 nmol C2 H4 /mg protein/day) followed by B. flexus (108.76 ± 3.66 nmol C2 H4 /mg protein/day) and B. circulans (98.28 ± 4.32 nmol C2 H4 /mg protein/day). Molecular basis of nitrogen fixation by these heterotrophic Bacillus strains has been explored in terms of the presence of nifH gene in them. We observed that heterotrophic Bacillus sp. have potential ability to fix nitrogen.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

Endomicrobium proavitum, the first isolate of Endomicrobia class. nov. (phylum Elusimicrobia)--an ultramicrobacterium with an unusual cell cycle that fixes nitrogen with a Group IV nitrogenase.

The bacterial tree contains many deep-rooting clades without any cultured representatives. One such clade is 'Endomicrobia', a class-level lineage in the phylum Elusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free-living member of this clade from sterile-filtered gut homogenate of defaunated (starch-fed) Reticulitermes santonensis. Strain Rsa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO2. Ultrastructural analysis revealed a Gram-negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of Group IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and (15)N2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to 'Candidatus Endomicrobium trichonymphae' and other obligate endosymbionts, we propose the name 'Endomicrobium proavitum' gen. nov., sp. nov. for the first isolate of this lineage and the name 'Endomicrobia' class. nov. for the entire clade.

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N2 -fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis-AAB association is consistent across years, we re-sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2 , we incubated twigs with (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N2 fixation in P. flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6 µg N m(-2)  d(-1) to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stable N2 -fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets.

Plant growth promoting potential and phylogenetic characteristics of a lichenized nitrogen fixing bacterium, Enterobacter cloacae.

Lichens are complex symbiotic association of mycobionts, photobionts, and bacteriobionts, including chemolithotropic bacteria. In the present study, 46 lichenized bacteria were isolated by conventional and enrichment culture methods on nitrogen-free bromothymol blue (NFb) medium. Only 11 of the 46 isolates fixed nitrogen on NFb and had reduced acetylene. All these 11 isolates had also produced siderophore and 10 of them the IAA. Further, ammonia production was recorded from nine of these nitrogen fixers (NF). On molecular characterization, 16 S rRNA sequencing recorded that, nine NF belonged to Proteobacteria, within Gammaproteobacteria, and were closely related to Enterobacter sp. with a maximum similarity to Enterobacter cloacae. Each one of our NF isolates was aligned closely to Enterobacter pulveris strain E443, Cronobacter sakazakii strain PNP8 and Providencia rettgeri strain ALK058. Notably, a few strains we examined found to possess plant growth promoting properties. This is the first report of Enterobacter sp. from lichens which may be inhabit lichen thalli extrinsically or intrinsically.

Isolation of N2 -fixing rhizobacteria from Lolium perenne and evaluating their plant growth promoting traits.

Twenty one dinitrogen (N2 )-fixing bacteria were isolated from the rhizosphere of Lolium perenne grown for more than 10 years without N-fertilization. The nearly complete sequence of the 16S rRNA gene of each strain and pairwise alignments among globally aligned sequences of the 16S rRNA genes clustered them into nine different groups. Out of the 21 strains, 11 were members of genus Bacillus, 3 belonged to each one of genera Paenibacillus and Pseudoxanthomonas, and the remaining 2 strains to each one of genera Burkholderia and Staphylococcus, respectively. A representative strain from each group contained the nifH gene and fixed atmospheric N2 as determined by the acetylene-dependent ethylene production assay (acetylene reduction activity, ARA). The nine selected strains were also examined to behave as plant growth promoting bacteria (PGPRs) including their ability to act as a biocontrol agent. The nine representative strains produced indol acetic acid (IAA) and solubilized calcium triphosphate, five of them, strains C2, C3, C12, C15, and C16, had ACC deaminase activity, and strains C2, C3, C4, C12, C16, and C17 produced siderophores. Strains C13, C16, and C17 had the capability to control growth of the pathogen Fusarium oxysporum mycelial growth in vitro. PCA analysis of determined PGPR properties showed that ARA, ACC deaminase activity, and siderophore production were the most valuable as they had the maximal contribution to the total variance.

A novel intracellular nitrogen-fixing symbiosis made by Ustilago maydis and Bacillus spp.

We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2 . Because only prokaryotic organisms have the capacity to reduce N2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of (15) N2 . Presence of an intracellular N2 -fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and (15) N incorporation into the cells proved that U. maydis fixed N2 . Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2 -fixing association involving a fungus and a bacterium.